Published December 5, 2014
Mammary tissue damage during bovine mastitis: Causes and control1
X. Zhao*2 and P. Lacasse†
*Department of Animal Science, McGill University, Ste. Anne de Bellevue, Québec, H9X 3V9, Canada;
and †Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada,
PO Box 90 STN Lennoxville, Sherbrooke, Quebec, J1M 1Z3, Canada
ABSTRACT: Mastitis, an inflammatory reaction of
the mammary gland that is usually caused by a micro-
bial infection, is recognized as the most costly disease
in dairy cattle. Decreased milk production accounts for
approximately 70% of the total cost of mastitis. Mam-
mary tissue damage reduces the number and activity
of epithelial cells and consequently contributes to de-
creased milk production. Mammary tissue damage has
been shown to be induced by either apoptosis or necro-
sis. These 2 distinct types of cell death can be distin-
guished by morphological, biochemical, and molecular
changes in dying cells. Both bacterial factors and host
immune reactions contribute to epithelial tissue dam-
age. During infection of the mammary glands, the tis-
sue damage can initially be caused by bacteria and their
products. Certain bacteria produce toxins that destroy
cell membranes and damage milk-producing tissue,
Key words: bovine, mammary gland, mastitis, epithelial cell, apoptosis, tissue damage
©2008 American Society of Animal Science. All rights reserved.
INTRODUCTION
Mastitis is defined as an inflammation of the mam-
mary gland. Mastitis usually occurs primarily in re-
sponse to intramammary bacterial infection, but also
to intramammary mycoplasmal, fungal, or algal infec-
tions. Mechanical trauma, thermal trauma, and chemi-
cal insult predispose the gland to intramammary infec-
tion (IMI). Occurrence of mastitis depends on the inter-
action of host, agent, and environmental factors.
Mastitis is the most costly infectious disease of dairy
cattle. The prevalence of mastitis in dairy cattle is rela-
tively high. Subclinical mastitis is the main form of
mastitis in modern dairy herds, exceeding 20 to 50% of
1
Presented at the Eighth International Workshop on the Biology
of Lactation in Farm Animals held in Pirassununga, Brazil, August
21–23, 2006.
2
Corresponding author: xin.zhao@mcgill.ca
Received May 25, 2007.
Accepted August 20, 2007.
57
whereas other bacteria are able to invade and multiply
within the bovine mammary epithelial cells before caus-
ing cell death. In addition, mastitis is characterized by
an influx of somatic cells, primarily polymorphonuclear
neutrophils, into the mammary gland. With more im-
mune cells migrating into the mammary gland and the
breakdown of the blood-milk barrier, damage to the
mammary epithelium worsens. It is well known that
breakdown of the extracellular matrix can lead to death
of the epithelial cells. Meanwhile, polymorphonuclear
neutrophils can harm the mammary tissue by releasing
reactive oxygen intermediates and proteolytic enzymes.
In vitro and in vivo studies suggest that the use of
antioxidants and other protective compounds in masti-
tis control programs is worth investigating, because
they may aid in alleviating damage to secretory cells
and thus reduce subsequent milk loss.
J. Anim. Sci. 2008. 86(Suppl. 1):57–65
doi:10.2527/jas.2007-0302
cows in given herds (Wilson et al., 1997; Pitkala et al.,
2004). The cost of subclinical mastitis is very difficult
to quantify, but most experts agree that subclinical
mastitis costs the average dairy farmer more than does
clinical mastitis. Assuming a 45% prevalence of subclin-
ical mastitis, the cost has been estimated in the range
of $180 to $320 per case (Wilson et al., 1997). Approxi-
mately 70% of this cost is associated with a reduction
in milk production. A large portion of it results from
irreversible damage to the mammary tissue (Oliver and
Calvinho, 1995). Although antibiotics are very useful
to treat the infection, they do not directly protect the
gland from being damaged.
This review is a compilation of some major findings
concerning tissue damage during mastitis. We describe
the current understanding of how bacteria, polymor-
phonuclear neutrophils (PMN), and host proteases and
cytokines contribute to tissue damage. Finally, research
aimed at the reduction of tissue damage is discussed.
OVERVIEW OF MASTITIS
Bovine mammary glands are exposed to diverse bac-
teria throughout lactation and in nonlactating periods.
58 Zhao and Lacasse
Pathogens commonly isolated from mastitic milk can
be classified as noncontagious (most are environmental)
and contagious microorganisms. The former include
Streptococcus uberis, Streptococcus dysgalactiae, Esche-
richia coli, and coagulase-negative staphylococcus spe-
cies, whereas the latter include Staphylococcus aureus
and Streptococcus agalactiae. The teat and streak canal
are the first line of defense of the mammary gland. The
keratin lining in the streak canal provides a physical
and chemical barrier against bacterial penetration (Ca-
puco et al., 1992). Bacteria may escape the natural
defense mechanisms by multiplication along the streak
canal (especially after milking), or by propulsion into
the teat cistern by vacuum fluctuations at the teat end
during milking. The infection occurs after bacteria gain
entrance to the mammary gland via the teat canal.
After bacteria overcome the anatomical defense, they
must evade the cellular and humoral defense mecha-
nisms of the mammary gland to establish disease (Sor-
dillo and Streicher, 2002). If the infection is not elimi-
nated, bacterial levels in the mammary gland eventu-
ally rise to a level at which they begin to damage the
mammary epithelium. As infection persists, the num-
ber of somatic cells in milk continues to increase and,
concomitantly, tissue damage is worsened. The alveoli
in the gland start to lose structural integrity and the
blood-milk barrier is breached. This allows extracellu-
lar fluid to enter the gland and mix with the milk.
Visible changes in the milk and the udder start to occur.
These can include external swelling, reddening of the
gland, and clotting and wateriness of the milk. By defi-
nition, this is the start of clinical symptoms. In brief,
bovine mammary epithelial cells can be damaged dur-
ing IMI by 1) release of a range of cellular and extracel-
lular products from bacterial pathogens; 2) lysosomal
enzymes and oxidative products released from phago-
cytes during phagocytosis of invading organisms, and
3) proteases from blood and cytokines released during
the immune response.
APOPTOSIS AND NECROSIS
Multiple-assay methodologies and different terminol-
ogies have been used by different research groups to
study and describe tissue damage during mastitis.
Thus, a brief introduction to types of cell death is war-
ranted. There are 2 distinct types of cell death,
apoptosis and necrosis, which may be distinguished by
morphological, biochemical, and molecular changes in
dying cells. Apoptosis is a process of deliberate suicide
of a cell in multicellular organisms. The process of
apoptosis was originally distinguished from necrosis on
the basis of cellular ultrastructure (Kerr et al., 1972).
Apoptosis may be identified by a characteristic pattern
of morphological changes, including nuclear and cyto-
plasmic condensation, nuclear fragmentation, and for-
mation of apoptotic bodies (Strange et al., 1992). These
changes are associated with cleavage of chromatin into
discretely sized oligonucleosome fragments by a cal-
cium-dependent endonuclease (Arends et al., 1990). Ap-
pearance of this oligonucleosomal DNA laddering in
stained gels is now widely used to detect apoptosis.
Enzymatic labeling of DNA strand breaks also allows
for histological detection of apoptosis in the tissue by a
terminal deoxynucleotidyl transferase-mediated dUTP
nick-end labeling (TUNEL) assay. In addition, loss of
asymmetry in cell membrane phospholipids during
apoptosis allows Annexin-V staining to be used to detect
exposed phosphatidylserine as a marker for apoptosis.
Furthermore, specific caspases, specific factors in apop-
totic pathways, and cleavage of caspase substrates have
been explored to detect apoptosis. Unlike apoptosis, ne-
crosis refers to the accidental death of cells in living
tissue. Necrosis may follow a wide variety of injuries,
both physical (e.g., cuts, burns, bruises) and biological
(e.g., effects of disease-causing agents). The sign of ne-
crosis, dead tissue, is called a lesion. It begins with
swelling of the cytoplasm and mitochondria caused by a
loss of membrane integrity and ends with total cell lysis.
These apparently clear-cut definitions between
apoptosis and necrosis, however, are likely oversimpli-
fied. For example, the apoptotic bodies as well as the
remaining cell fragments, if not quickly removed by
phagocytes or neighboring cells, will ultimately swell
and finally lyse. This process is called secondary necro-
sis. In addition, the positive or negative interconnection
between autophagy and apoptosis (Gozuacik and Kim-
chi, 2004) adds additional complexity to apoptosis re-
search.
Cytotoxicity is also commonly used in the literature
to describe tissue damage, but does not infer a specific
cellular mechanism. Rather, it indicates the cell-killing
property of a chemical compound or a mediator cell.
Many assays are available for measuring cytotoxicity,
and most of them are based on increased plasma mem-
brane permeability or increased uptake of dyes by dying
cells or dead cells. The results from cytotoxicity assays
vary considerably depending on the method used (Fel-
lows and O’Donovan 2007). In general, cytotoxicity
assays can be used to detect necrosis and the late stage
of apoptosis, but not the early stage of apoptosis. Spe-
cifically for bovine mammary studies, lactate dehydro-
genase activity and N-acetyl-β-D-glucosaminidase
(NAGase) activity have commonly been used as mark-
ers for tissue damage. The former enzyme is a stable
cytoplasmic enzyme present in all cells. It is rapidly
released outside the cell when the plasma membrane
is damaged. Located within lysosomes of mammary epi-
thelial cells, NAGase is released from damaged mam-
mary epithelial cells (Kitchen et al., 1980). However,
a small fraction of milk NAGase can also come from
leukocytes (Capuco et al., 1986).
TISSUE DAMAGE: HISTOLOGICAL
OBSERVATION
Histological analyses have been widely used since
the 1970s and are still being used today for assessing
 Tissue damage and mastitis
damage to secretory tissue in the bovine mammary
gland caused by mastitis pathogens. For example, Be-
nites et al. (2002) examined the mammary parenchyma
from 184 slaughtered dairy cows for the existence of
microorganisms and histopathological changes. Of all
the samples from which microorganisms were isolated,
only 3.1% did not show histological changes. The re-
maining 96.9% of samples showed an inflammatory re-
sponse (i.e., edema, mammary epithelial cell damage,
and PMN infiltration), tissue repair process, or both.
In contrast, 33.9% of samples from glands without evi-
dence of microorganisms (n = 56) showed no histological
changes. These results clearly indicate that the pres-
ence of microorganisms is associated with tissue
damage.
Escherichia coli is one of the most important patho-
gens causing mastitis in dairy cows (Bradley, 2002),
resulting in inflammation that ranges from subacute to
peracute. Necrosis of the mammary epithelium occurs
during severe, naturally occurring clinical E. coli masti-
tis, as well as during severe experimental E. coli masti-
tis. In moderate cases of E. coli mastitis, there is mini-
mal alveolar tissue damage, as shown by Frost et al.
(1980). In that experiment, the main changes were su-
perficial and confined to the epithelium of the teat sinus,
lactiferous sinus, and large ducts, without serious
involvement of the secretory tissue. There were some
lesions of the basement membrane underlying the dam-
aged epithelium but damage was localized. In very se-
vere cases, the infection progressed via the ductile sys-
tem to produce a limited inflammatory reaction but
with an extensive involvement of the secretory tissue
(Frost and Brooker, 1986). In its most severe form with
uncontrolled bacterial multiplication, all lactiferous si-
nus epithelia were lost, interstitial tissue became hem-
orrhagic, and often the animal died of toxemia within
a few hours of infection (Burvenich et al., 2003). The
severity of the disease varies considerably from cow to
cow. Kornalijnslijper et al. (2004) reported that 6 h after
intramammary inoculation, the bacterial counts, which
were significantly correlated with severity of the dis-
ease, varied considerably (i.e., up to 800 fold). They
concluded that bacterial growth in the phase before
massive influx of PMN is a major determinant in the
final severity of experimental E. coli mastitis. To deter-
mine whether the endotoxin lipopolysaccharide (LPS)
is responsible for E. coli-mediated tissue damage, Ca-
puco et al. (1985) cultured lactating mammary tissue
explants in the presence of E. coli endotoxin and a labo-
ratory culture filtrate of E. coli. Results indicated that
endotoxin had no effect, but the culture filtrate dam-
aged tissue and decreased milk synthesis and secretion.
One of the most common types of chronic mastitis is
caused by Staph. aureus. Histopathological responses of
lactating tissue to experimental or naturally occurring
Staph. aureus mastitis were extensively studied during
the 1970s and 1980s. Chandler and Reid (1973) exam-
ined mammary parenchymal tissue samples from lac-
tating cows infected naturally with Staph. aureus and
59
reported a massive PMN infiltration and necrosis of
secretory tissues. In addition, Heald (1979) observed
that mammary tissues from lactating cows inoculated
with Staph. aureus exhibited less milk synthesis and
secretion, as evidenced by more interalveolar stroma
and involuting alveolar epithelium and less alveolar
luminal space compared with uninfected contralateral
controls. Moreover, these changes were associated with
replacement of secretory tissue with nonsecretory tis-
sue (Nickerson and Heald, 1981). Similarly, a study
with dry cows revealed that quarters with intramam-
mary Staph. aureus infection had a greater degree of
mammary involution with a greater proportion of inter-
alveolar stroma and a lesser proportion of alveolar lu-
men compared with uninfected quarters (Sordillo and
Nickerson, 1988). Finally, a challenge study with heif-
ers by Trinidad et al. (1990) showed that mammary
glands infected by Staph. aureus also exhibited fewer
alveolar epithelial and luminal areas but more interal-
veolar stroma than controls. From these results, we are
relatively confident in concluding that Staph. aureus
infection causes necrosis of the secretory tissues and
that the damaged secretory tissue is replaced with non-
secretory tissue.
APOPTOSIS DURING MASTITIS
The induction of apoptosis by bacterial pathogens
is a well-established cellular process (Weinrauch and
Zychlinsky, 1999). Apoptosis is a key feature of mam-
mary gland development and function. In addition,
apoptosis is critical for removing the milk-secreting al-
veolar epithelial cells during lactation and postlacta-
tional involution (Capuco and Akers, 1999; Capuco et
al., 2003). However, conclusive and direct evidence for
involvement of apoptosis during mastitis has been pro-
vided only by Long et al. (2001). Escherichia coli-in-
fected mammary glands were biopsied, with the re-
sulting tissues processed for RNA, protein, and histo-
logical examinations. Both mRNA and protein analyses
indicated up-regulation of the proapoptotic factors Bax
and IL-1β-converting enzyme, and a down-regulation
of the antiapoptotic factor Bcl-2. Further, induction of
a 92-kDa gelatinase was observed by gelatin zymogra-
phy. Finally, the number of apoptotic epithelial cells per
10 microscopic sections, as determined by the TUNEL
assay, increased from 1.8 ± 0.5 to 8.8 ± 2.8 cells. In
addition, there was evidence for induction of apoptosis
by other mastitis pathogens. For example, Sheffield
(1997) reported a 5-fold induction of a putative marker
of apoptosis, testosterone-repressed prostate mucin-2
mRNA, after experimental infection of the bovine mam-
mary gland with Strep. agalactiae. Finally, in vitro
studies indicated that Staph. aureus caused apoptosis
in a bovine mammary cell line (Bayles et al., 1998).
Nevertheless, the effect of Staph. aureus and Strep.
agalactiae on apoptosis of mammary tissue still needs
to be confirmed in vivo. It is worthwhile to indicate
that apoptosis does not cause major histomorphological
60 Zhao and Lacasse
changes, as observed in traditional mastitis studies;
however, it does reduce cell numbers in mammary tis-
sue. In addition, apoptosis may cause tissue damage in
a delayed fashion through secondary necrosis (Medan
et al., 2002).
The clearance processes of apoptotic cells during mas-
titis are not totally understood. The clearance of apop-
totic cells by phagocytes, such as macrophages, has
been shown. In addition, mammary epithelial cells are
known to be capable of phagocytosing apoptotic cells in
species other than cattle (Monks et al., 2002). Because
methods for detection of apoptosis reveal only the cells
involved in the process, it is quite possible that
apoptosis during mastitis is underestimated.
INVOLVEMENT OF NEUTROPHILS
IN TISSUE DAMAGE
Following detection of pathogen invasion into the
mammary gland, macrophages and epithelial cells re-
lease chemoattractants. These agents trigger the mi-
gration of leukocytes, mainly PMN, from the blood to-
ward the mammary gland and increase their propor-
tions from a basal level of 5 to 25% to approximately
90% of total cells in the milk (Leitner et al., 2000; Riollet
et al., 2000a). These PMN are considered as the second
line of defense of the mammary gland. The presence of
functional PMN is crucial to the host defense against
bacterial pathogens (Paape et al., 2003).
The main functions of PMN are to engulf pathogens
and destroy them via oxygen-dependent and oxygen-
independent systems. At the same time, PMN can po-
tentially harm the mammary gland. The exact mecha-
nism by which PMN damage bovine epithelial cells dur-
ing mastitis is still not fully understood. Neutrophils
may promote tissue injury and disturb mammary func-
tion, via reactive oxygen metabolite generation (i.e., the
respiratory burst) and granular enzyme release (i.e.,
degranulation; Paape et al., 2002).
During experimentally induced Staph. aureus masti-
tis, migration of PMN through the secretory epithelium
was correlated with extensive morphological damage
(Harmon and Heald, 1982). Direct evidence that PMN
damage mammary tissue was provided by Capuco et
al. (1986). Neutrophils isolated from mammary glands
of nulliparous heifers given an injection of E. coli endo-
toxin were incubated with mammary tissues from non-
infected quarters. Microscopic examination indicated
that epithelial cell damage resulted from treatment
with intact, lysed, or phagocytosing PMN. The greatest
morphological damage resulted from treatment with
phagocytosing PMN. This observation was confirmed
in an in vitro coculture system with mammary epithe-
lial cells and PMN. Activated blood PMN were cytotoxic
for mammary epithelial cells (Ledbetter et al., 2001;
Lauzon et al., 2005), possibly via the release of extracel-
lular reactive oxygen species, such as hydroxyl radicals
(Boulanger et al., 2002). Oxidative stress can damage
all types of biomolecules (e.g., DNA, proteins, lipids,
and carbohydrates) and therefore induce tissue injury.
Bovine PMN have primary (azurophilic), secondary,
and tertiary granules (Paape et al., 2002, 2003). These
intracellular granules contain bactericidal peptides,
proteins, and enzymes such as elastase, other protein-
ases, and myeloperoxidase that are released into phago-
cytic vacuoles or the extracellular environment
(Faurschou and Borregaard, 2003). Proteolytic enzymes
in PMN include neutral and acidic proteases. Elastase
(EC 3.4.21.36) and cathepsin G (EC 3.4.21.20) are the
predominant enzymes in PMN (Bank and Ansorge,
2001). Other proteases in PMN include the thiol prote-
ase cathepsin B (EC 3.4.22.1) and the acid protease
cathepsin D (EC 3.4.23.5), as reported by Owen and
Campbell (1999). Furthermore, activated bovine PMN
can express matrix metalloproteinase (MMP)-9 (Li et
al., 1999).
Milk PMN usually have lower total proteolytic enzy-
matic activities than peripheral blood PMN (Prin-Ma-
thieu et al., 2002), indicating that PMN release many
proteases during migration into the mammary gland.
Using an E. coli mastitis model, Haddadi et al. (2006)
observed that milk PMN had lower cathepsin and colla-
genase activities than peripheral blood PMN. Thus,
PMN appear to use up part of these enzymes during
their migration to cross the endothelium, extracellular
matrix (ECM), and epithelium (Faurschou and Borreg-
aard, 2003). In contrast, Le Roux et al. (2003) reported
that elastase was higher in milk than in blood. Elastase
is localized in azurophilic granules of PMN, and its
increase in milk is probably due to proinflammatory
cytokines such as IL-6, IL-2, and tumor necrosis factor-
α, which increase the transcription of serine proteases,
including elastase and cathepsin G (Le Roux et al.,
2003). Thus, the amount and variety of enzymes re-
leased by PMN into milk seem to be affected by the
migration and activation of PMN.
The enzymes involved in bovine mammary tissue de-
struction were investigated by Mehrzad et al. (2005)
by using an endotoxin-induced mastitis model. They
reported that mastitic milk proteases hydrolyzed ca-
sein, gelatin, collagen, hemoglobin, mammary gland
membrane proteins, and lactoferrin, indicating that
mastitic milk proteases have a broad spectrum of activi-
ties. Further, the direct involvement of proteases in
epithelial cell damage was demonstrated by the fact
that coincubation of normal mammary tissue with mas-
titic milk, but not normal milk, caused tissue degrada-
tion (Mehrzad et al., 2005). Therefore, proteases re-
leased by PMN are likely involved in mammary tissue
damage during mastitis.
It is unlikely that the diapedesis process itself is di-
rectly involved in epithelial damage. Under normal con-
ditions, PMN can migrate into mammary glands with-
out damaging the tissue. Further, PMN diapedesis it-
self did not cause detectable epithelial cell damage in
an in vitro study (Lin et al., 1995). However, prolonged
diapedesis of leukocytes could cause damage to mam-
 Tissue damage and mastitis
mary parenchymal tissue, resulting in decreased pro-
duction of milk (Harmon and Heald, 1982; Sordillo and
Nickerson, 1988). This damage may occur through sev-
eral mechanisms, including premature activation dur-
ing migration, extracellular release of toxic products
during the killing of some microbes, removal of infected
or damaged host cells and debris as a first step in tissue
remodeling, or failure to terminate acute inflamma-
tory responses.
The PMN-induced damage is probably somewhat con-
tained. After ingestion and release of their chemicals,
most of the milk PMN perish by induction of apoptosis.
This is followed by the migration of macrophages into
the mammary gland and engulfment of PMN by these
macrophages. Through this process, damaging chemi-
cals are walled off within dying PMN, which are then
ingested by macrophages to minimize further damage
to tissue (Paape et al., 2002, 2003).
INVOLVEMENT OF BACTERIA
IN TISSUE DAMAGE
There is increasing evidence that pathogens use vari-
ous mechanisms to impinge upon cell death pathways.
A number of pathogens are armed with an array of
virulence determinants, which interact with key compo-
nents of a host cell’s death pathways or interfere with
regulation of transcription factors monitoring cell sur-
vival. These virulence factors induce cell death by a
variety of mechanisms, which include 1) pore-forming
toxins, which interact with the host cell membrane and
permit the leakage of cellular components; 2) toxins
that express their enzymatic activity in the host cytosol;
3) effector proteins delivered directly into host cells
by a highly specialized type-III secretory system; 4)
superantigens that target immune cells, and 5) other
modulators of host cell death (Weinrauch and Zychlin-
sky, 1999). Much progress has been made in under-
standing the role of apoptosis and necrosis in response
to bacterial infection.
Escherichia coli produces a number of proteinases,
including collagenolytic enzymes, which contribute to
the degradation of ECM components (Haddadi et al.,
2005, 2006). There is no doubt that these proteolytic
activities contribute to the apoptosis and necrosis of
mammary epithelial cells. However, the degree to
which they contribute is still unknown.
Endotoxin LPS is considered a key factor for the
pathogenicity of gram-negative bacteria. Accordingly,
intramammary infusion of E. coli LPS is often used to
study events occurring during E. coli mastitis, because
it mimics the symptoms of naturally occurring mastitis
without microorganism invasion and toxin production,
which could cause direct damaging effects on the mam-
mary epithelial cells (Oliver and Calvinho, 1995). When
LPS was used to induce an inflammatory response in
the mammary gland, no lesions were observed in the
mammary glands (Hill, 1991). Similarly, LPS does not
cause tissue damage in vitro in explants of lactating
61
bovine mammary tissue (Capuco et al., 1985) or mam-
mary epithelial cells (Boulanger et al., 2002). However,
the possibility for involvement of LPS in E. coli-induced
cell damage cannot be totally ruled out, because LPS
stimulated the expression of IL-1 by MAC-T cells, an
established bovine mammary epithelial cell line (Boud-
jellab et al., 2000). In addition, LPS increased expres-
sion of urokinase-type plasminogen activator (uPA;
Ohta et al., 2000). Therefore, it is plausible that LPS
induces apoptosis or necrosis in mammary epithelial
cells indirectly through induction of proteases or proin-
flammatory cytokines. At this time, however, there is
no experimental evidence to support this notion, pre-
sumably because of the previous lack of sensitive ana-
lytical methods.
Staphylococcus aureus produces toxins that destroy
cell membranes, directly damage milk-producing tis-
sue, and induce necrosis in bovine mammary glands
(Chandler and Reid, 1973; Heald, 1979; Nickerson and
Heald, 1981; Sordillo and Nickerson, 1988; Trinidad et
al., 1990). Initially, the bacteria damage tissues lining
the teat and gland cisterns within the quarter. If un-
checked, they invade the duct system and establish
deep-seated pockets of infection in the milk-secreting
cells (i.e., alveoli). This is followed by the walling-off of
bacteria by scar tissue and the formation of abscesses.
Moreover, Bayles et al. (1998) provided evidence that
Staph. aureus induced apoptosis in bovine mammary
gland epithelial cells. They showed that 2 h after the
internalization of one Staph. aureus mastitis isolate,
MAC-T cells exhibited detachment from the matrix,
rounding, a mottled cell membrane, and vacuolation
of the cytoplasm, all of which are indicative of cells
undergoing apoptosis. By 18 h, the majority of the MAC-
T cell population exhibited an apoptotic morphology.
Other evidence for apoptosis was the existence of a
characteristic DNA ladder pattern and the positive TU-
NEL labeling of apoptotic cells. These results clearly
demonstrate that after internalization, Staph. aureus
escapes the endosome and induces apoptosis in mam-
mary epithelial cells. The apoptosis depends on factors
regulated by the global virulence gene regulators Agr
and Sar (Wesson et al., 1998), as well as by caspases 8
and 3 (Wesson et al., 2000). Although there is still no
direct evidence showing that Staph. aureus induces
apoptosis of bovine mammary epithelial cells in vivo,
this seems likely.
Staphylococcus aureus produces a wide variety of exo-
proteins that contribute to its ability to colonize and
cause disease in mammalian hosts. Nearly all strains
examined secrete a group of enzymes and cytotoxins,
which includes 4 hemolysins (α, β, λ, and δ), nucleases,
proteases, lipases, hyaluronidase, and collagenase. The
main function of these proteins may be to convert local
host tissues into the nutrients required for bacterial
growth. Some strains produce one or more additional
exoproteins, which include toxic shock syndrome toxin-
1, staphylococcal enterotoxins, exfoliative toxins, and
leukocidin (Dinges et al., 2000). Strains isolated from
62 Zhao and Lacasse
cases of bovine mastitis express α, β, λ, and δ toxins,
leukocidins, enterotoxin, and coagulase (Bramley et al.,
1989; Matsunaga et al., 1993). Among all the Staph.
aureus cytotoxins, α-hemolysin has been the most care-
fully examined. It can induce both apoptosis and necro-
sis in eukaryotic cells, depending on the dosage given
(reviewed by Weinrauch and Zychlinsky, 1999). At low
doses, the toxin binds to specific, but as yet unidentified,
cell surface receptors and produces small pores that
selectively facilitate the release of monovalent ions, re-
sulting in characteristic DNA fragmentation and cell
death. At high doses, α-hemolysin nonspecifically ab-
sorbs to the lipid bilayer and forms larger pores in the
membrane that are Ca2+ permissive, which results in
massive necrosis without DNA fragmentation (Wein-
rauch and Zychlinsky, 1999). Whether this also applies
to bovine mammary epithelial cells is unknown. Other
bacterial toxins could also have damaging effects on
bovine mammary tissues, but they have not been stud-
ied intensively. Kuroishi et al. (2003) inoculated mam-
mary glands with staphylococcal enterotoxin C and ob-
served epithelial cellular degeneration, including inva-
gination and cytoplasmic vacuolation of epithelial cells.
It is not clear whether this effect is due to increased
production of the superoxide by migrated PMN or
whether it is a direct effect of staphylococcal enterotoxin
C on mammary epithelial cells.
Streptococcus dysgalactiae is an environmental
pathogen that causes bovine IMI. Almeida and Oliver
(1995) assayed 2 mastitis strains of Strep. dysgalactiae
for their ability to invade, multiply, and induce damage
to MAC-T cells. They demonstrated that the invasion
process did not appear to affect the viability of mam-
mary epithelial cells but that cellular damage was in-
duced, as indicated by the release of increasing amounts
of lactate dehydrogenase (Almeida and Oliver, 1995).
INVOLVEMENT OF PLASMA PROTEINS
AND CYTOKINES IN TISSUE DAMAGE
At the onset of mastitis, increased permeability of
the blood-milk mammary epithelial barrier leads first
to an influx of serum constituents, such as plasminogen
and numerous other enzymes, and second to a massive
recruitment of somatic cells, in particular, PMN (Le
Roux et al., 2003). Milk contains 2 proteinase systems
derived from blood, one of which is involved in dissolv-
ing blood clots (i.e., plasmin) and the other in defense
against invasive microorganisms (i.e., lysosomal pro-
teinases of somatic cells). Whereas plasmin is the prin-
cipal proteinase in good-quality milk, other proteinases,
including cathepsins and elastase, are probably also
active, particularly as the somatic cell count of milk
increases (Kelly et al., 2006; Leitner et al., 2006). This
is supported by the observation that the protease activi-
ties of plasmin and mastitic milk differ (Mehrzad et
al., 2005). In addition, mammary epithelial cells also
express certain MMP and serine proteases, which are
involved in the activation of plasminogen to plasmin
(Green and Lund, 2005).
The plasmin-plasminogen activator system in bovine
milk is closely correlated with gradual involution (Pol-
itis, 1996). Plasmin directly degrades matrix proteins
such as fibrin and laminin and also activates MMP
precursors, such as pro-MMP-3, MMP-9, and MMP-
13 (Green and Lund, 2005). The increase in plasmin
activity during mastitis is linked to the permeability
of the milk epithelial barrier during inflammation. In
addition, numerous activators of plasmin and its zymo-
gen come from the bloodstream, PMN, and bacteria.
During mastitis, the serine protease activator concen-
tration, including those for plasmin and plasminogen
in blood and also in milk, increases sharply (Le Roux
et al., 2003; Moussaoui et al., 2004). Polymorphonuclear
neutrophils have a pool of plasminogen activators, such
as uPA and, to a lesser extent, tissue plasminogen acti-
vators (Moir et al., 2001). Several bacteria, such as
Staph. aureus, E. coli, and Salmonella typhimurium,
express a plasminogen receptor on their surfaces,
which, through immobilization of plasminogen, en-
hances plasminogen activation into plasmin (Lahteen-
maki et al., 2001).
How much plasmin contributes to tissue damage dur-
ing mastitis is debatable. Mammary epithelial cell via-
bility depends on attachment to the ECM, so it is rea-
sonable to postulate that ECM degradation is involved
in tissue damage and cell death during mastitis. Ex-
pression of MMP-9, stromelysin-1 mRNA, and uPA
mRNA was increased in association with apoptosis dur-
ing E. coli mastitis (Long et al., 2001). Matrix metallo-
protease-9 can be produced by bovine PMN (Li et al.,
1999) and MAC-T cells (Long et al., 2001). Stromelysin-
1 contributed to the breakdown of most ECM compo-
nents, including laminin and collagen type IV (Rudolph-
Owen and Matrisian, 1998). Other proteases that have
been reported to increase in milk from mastitic cows
include MMP-2 and MMP-9 (Raulo et al., 2002; Lauzon
et al., 2006), as well as a 120-kDa gelatinase (Mehrzad
et al., 2005; Lauzon et al., 2006). Mehrzad et al. (2005)
also tested proteolytic activity directly in normal mam-
mary tissue of mastitic cows. They found that lac-
toserum from mastitic cows was more proteolytic than
normal lactoserum. Lactoserum from mastitic cows ex-
foliated the cells and surrounding proteins, leaving a
nude dense collagen network. These differences re-
sulted from protease contents and activities, which
were significantly higher in mastitic milk (Mehrzad et
al., 2005). They concluded that the proteolysis and tis-
sue damage observed were largely due to MMP. How-
ever, the possible contribution from other nonplasmin
proteases and plasmin should not be ignored.
Cytokines are central mediators of inflammatory
events during IMI. Bacteria release potent toxins that
trigger white blood cells and epithelial cells in the mam-
mary gland to secrete cytokines (Paape et al., 2003).
Intramammary challenge with E. coli or Staph. aureus
has elicited differential innate immune responses in
 Tissue damage and mastitis
terms of clinical symptoms and milk cytokine profiles
at the protein level (Bannerman et al., 2004; Riollet et
al., 2000b) and at mRNA levels (Lee et al., 2006). The
difference in cytokine profiles may underlie the differ-
ences in the sequela of symptoms for these 2 types
of mastitis.
Very little work has been conducted to determine the
role of cytokines in the regulation of tissue damage
during mastitis. Cytokines recruit PMN that function
as phagocytes at the site of infection. During a study
on the use of recombinant bovine (rb) IL-8 as a potential
therapeutic agent for subclinical mastitis of dairy cows,
Takahashi et al. (2005) observed significant increases
in milk somatic cell counts and in chemiluminescence
activity after intramammary injection of rbIL-8. Al-
though they did not study the tissue damage, the high
levels of milk somatic cell counts and free radical pro-
duction would certainly have led to tissue damage. It
has previously been reported that rbIL-8 can induce
the migration of PMN through an in vitro model of the
endo-epithelial barrier consisting of the bovine aortic
endothelial cell line and MAC-T cells (Lee and Zhao,
2000). Other cytokines, such as tumor necrosis factor-
α and IL-1, induce apoptosis in a variety of cell types,
including bovine endothelial cells (Mebmer et al., 1999)
and human mammary epithelial cells (Burow et al.,
1999). Levels of these cytokines increase during E. coli
mastitis (Bannerman et al., 2004), and it is tempting
to assume that they also induce apoptosis in bovine
mammary epithelial cells. A range of cytokines are also
known to promote a wide variety of functions of PMN,
including adhesion, surface receptor expression, free
radical production, and release of lysosomal constit-
uents (Paape et al., 2003). Therefore, the effects of cy-
tokines on tissue damage are more likely to be mediated
through recruitment and activation of PMN.
REDUCTION OF TISSUE DAMAGE
Although the mechanisms underlying tissue damage
during mastitis have not been fully delineated, there
are likely opportunities for pharmacological interven-
tion to block the proteolytic or oxidative cascade within
the inflamed gland to reduce tissue damage during mas-
titis. Up to now, most work in this area has been focused
on the reduction of free radicals, because results from
research using in vitro models indicate that LPS stimu-
lates PMN-induced damage to mammary epithelial
cells by producing superoxide (Boulanger et al., 2002).
The major free radicals found in biological systems
are superoxide, hydrogen peroxide, hydroxyl radical,
and fatty acid radicals (Ward et al., 1988). Hydrogen
peroxide is found primarily in the cytosol of cells, and
fatty acid radicals are found primarily in cell mem-
branes (Ward et al., 1988). Superoxide and hydroxyl
radicals can be found in both cell components (Paape
et al., 2003). Because free radicals are extremely toxic
to cells, the body has developed a sophisticated antioxi-
dant system. Superoxide dismutase converts superox-
63
ide to hydrogen peroxide. Hydrogen peroxide is con-
verted to water by the enzyme glutathione peroxidase
(Ward et al., 1988). Those 2 enzymes effectively control
most free radicals within the cytosol. Accordingly, the
use of antioxidants to prevent oxidative stress has been
studied in several types of inflammation (Cuzzocrea et
al., 2004).
Using a coculture model of activated bovine PMN
and MAC-T cells, Lauzon et al. (2005) found that 3
antioxidants (i.e., catechin, deferoxamine, or glutathi-
one ethyl ester) could partially or totally eliminate the
damaging effect of activated bovine PMN to MAC-T
cells, indicating that antioxidants may be effective tools
for protecting mammary tissue against PMN-induced
oxidative stress during bovine mastitis. The protective
effects of these 3 antioxidants on PMN-induced damage
to mammary cells were further evaluated in vivo by
using an endotoxin-induced mastitis model. The extent
of cell damage was evaluated by measuring milk levels
of lactate dehydrogenase and NAGase activity. Intra-
mammary infusions of catechin or glutathione ethyl
ester did not exert any protective effect, whereas infu-
sion of deferoxamine, a chelator of iron, decreased milk
lactate dehydrogenase and NAGase activity, indicating
a protective effect against PMN-induced damage. The
protective effect of deferoxamine was also evident from
a lower level of haptoglobin in milk. The proteolytic
activity of mastitic milk was not influenced by the pres-
ence of deferoxamine. Overall, these results indicate
that local infusion of deferoxamine may be an effective
tool to protect mammary tissue against PMN-induced
oxidative stress during bovine mastitis (Lauzon et
al., 2006).
SUMMARY
Despite considerable advances in the understanding
of the pathogenesis of bovine mastitis, the disease con-
tinues to be an economically important problem in the
dairy industry throughout the world. The ideal means
of dealing with mastitis is to prevent it from occurring.
However, even under the best prevention and control
programs, mastitis will still occur. Reducing economic
losses resulting from mastitis is still a daunting chal-
lenge. Although it is accepted that neutrophils, bacte-
ria, and host proteases and cytokines contribute to
mammary tissue damage during mastitis, many finer
details are still unknown. Mammary tissue damage
during IMI is probably underestimated because of the
lack of sensitive and noninvasive detection methods.
Early detection of IMI is a prerequisite for initiating
any intervention measure to minimize the mammary
tissue damage. The mechanisms responsible for mam-
mary epithelium and tissue damage during mastitis
are still not well known, but they include both apoptosis
and necrosis. Understanding the biochemical and cellu-
lar changes that occur in the gland during mastitis will
ultimately lead to a means of manipulating mammary
functions to minimize the damage from mastitis. In
64 Zhao and Lacasse
addition to the use of antibiotics to treat mastitis, other
measures for reducing tissue damage may be a cost-
effective way to reduce the losses caused by mastitis.
LITERATURE CITED
Almeida, R. A., and S. P. Oliver. 1995. Invasion of bovine mammary
epithelial cells by Streptococcus dysgalactiae. J. Dairy Sci.
78:1310–1317.
Arends, M. J., R. G. Morris, and A. H. Wyllie. 1990. Apoptosis: The
role of endonuclease. Am. J. Pathol. 136:593–608.
Bank, U., and S. Ansorge. 2001. More than destructive: Neutrophil
derived serine proteases in cytokine bioactivity control. J. Leu-
koc. Biol. 69:197–206.
Bannerman, D. D., M. J. Paape, J. W. Lee, X. Zhao, J. C. Hope, and
P. Rainard. 2004. Escherichia coli and Staphylococcus aureus
elicit different innate immune responses following intramam-
mary infection. Clin. Diagn. Lab. Immunol. 11:463–472.
Bayles, K. W., C. A. Wesson, L. E. Liou, L. K. Fox, G. A. Bohach,
and W. R. Trumble. 1998. Intracellular Staphylococcus aureus
escapes the endosome and induces apoptosis in epithelial cells.
Infect. Immun. 66:336–342.
Benites, N. R., J. L. Guerra, P. A. Melville, and E. O. da Costa. 2002.
Aetiology and histopathology of bovine mastitis of a spontaneous
occurrence. J. Vet. Med. B 49:366–370.
Boudjellab, N., H. S. Chan-Tang, and X. Zhao. 2000. Bovine interleu-
kin-1 expression by cultured mammary epithelial cells (MAC-
T) and its involvement in the release of MAC-T derived interleu-
kin-8. Comp. Physiol. Biochem. 127:191–199.
Boulanger, V., X. Zhao, and P. Lacasse. 2002. Protective effect of
melatonin and catalase in bovine neutrophil-induced model of
mammary cell damage. J. Dairy Sci. 85:562–569.
Bradley, A. J. 2002. Bovine mastitis: An evolving disease. Vet. J.
163:1–13.
Bramley, A. J., A. H. Patel, M. O’Reilly, R. Foster, and T. J. Foster.
1989. Roles of alpha-toxin and beta-toxin in virulence of Staphy-
lococcus aureus for the mouse mammary gland. Infect. Immun.
57:2489–2494.
Burow, M. E., Y. Tang, B. M. Collins-Burow, S. Krajewski, J. C.
Reed, J. A. McLachlan, and B. S. Bechman. 1999. Effects of
environmental estrogens on tumor necrosis factor α-mediated
apoptosis in MCF-7 cells. Carcinogenesis 20:2057–2061.
Burvenich, C., V. van Merris, J. Mehrzad, A. Diez-Fraile, and L.
Duchateau. 2003. Severity of E. coli mastitis is mainly deter-
mined by cow factors. Vet. Res. 34:521–564.
Capuco, A. V., and R. M. Akers. 1999. Mammary involution in dairy
animals. J. Mammary Gland Biol. Neoplasia 4:137–144.
Capuco, A. V., S. A. Bright, J. W. Pankey, D. L. Wood, R. H. Miller,
and J. Bitman. 1992. Increased susceptibility to intramammary
infection following removal of teat canal keratin. J. Dairy Sci.
75:2126–2130.
Capuco, A. V., S. E. Ellis, S. A. Hale, E. Long, R. A. Erdman, X. Zhao,
and M. J. Paape. 2003. Lactation persistency: Insights from
mammary cell proliferation studies. J. Anim. Sci. 81(Suppl.
3):18–31.
Capuco, A. V., M. J. Paape, and S. C. Nickerson. 1986. In vitro study
of polymorphonuclear leukocyte damage to mammary tissues of
lactating cows. Am. J. Vet. Res. 47:663–668.
Capuco, A. V., M. J. Paape, J. J. Smith, and D. A. Loeffler. 1985. In
vitro effect of bacterial toxins on lactating bovine mammary
tissue. J. Dairy Sci. 68(Suppl. 1):206.
Chandler, R. L., and I. M. Reid. 1973. Ultrastructure and associated
observations in clinical cases of mastitis in cattle. J. Comp. Pa-
thol. 83:233–241.
Cuzzocrea, S., C. Thiemermann, and D. Salvemini. 2004. Potential
therapeutic effect of antioxidant therapy in shock and inflam-
mation. Curr. Med. Chem. 11:1147–1162.
Dinges, M. M., P. M. Orwin, and P. M. Schlievert. 2000. Exotoxins
of Staphylococcus aureus. Clin. Microbiol. Rev. 13:16–34.
Faurschou, M., and N. Borregaard. 2003. Neutrophil granules and
secretory vesicles in inflammation. Microb. Infect. 5:1317–1327.
Fellows, M. D., and M. R. O’Donovan. 2007. Cytotoxicity in cultured
mammalian cells is a function of the method used to estimate
it. Mutagenesis 22:275–280.
Frost, A. J., and B. E. Brooker. 1986. Hyperacute Escherichia coli
mastitis of cattle in the immediate post-partum period. Aust.
Vet. J. 63:327–331.
Frost, A. J., A. W. Hill, and B. E. Brooker. 1980. The early pathogene-
sis of bovine mastitis due to Escherichia coli. Proc. R. Soc. Lond.
B. Biol. Sci. 209:419–431.
Gozuacik, D., and A. Kimchi. 2004. Autophagy as a cell death and
tumor suppressor mechanism. Oncogene 23:2891–2906.
Green, K. A., and L. R. Lund. 2005. ECM degrading proteases and
tissue remodeling in the mammary gland. Bioessays 27:894–903.
Haddadi, K., F. Moussaoui, I. Hebia, F. Laurent, and Y. Le Roux.
2005. E. coli proteolytic activity in milk and casein breakdown.
Reprod. Nutr. Dev. 45:485–496.
Haddadi, K., C. Prin-Mathieu, F. Moussaoui, G. C. Faure, F. Van-
groenweghe, C. Burvenich, and Y. Le Roux. 2006. Polymorpho-
nuclear neutrophils and Escherichia coli proteases involved in
proteolysis of casein during experimental E. coli mastitis. Int.
Dairy J. 16:639–647.
Harmon, R. J., and C. W. Heald. 1982. Migration of polymorphonu-
clear leukocytes into the bovine mammary gland during experi-
mentally induced Staphylococcus aureus mastitis. Am. J. Vet.
Res. 43:992–998.
Heald, C. W. 1979. Morphometric study of experimentally induced
Staphylococcus aureus mastitis in the cow. Am. J. Vet. Res.
40:1294–1298.
Hill, A. W. 1991. Somatic cells—Friends or foes? Pages 217–232 in
New Insights into the Pathogenesis of Mastitis. C. Burvenich,
G. Vandeputte-Van Messom, and A. W. Hill, ed. Flemish Vet.
J., Merelbeke, Belgium.
Kelly, A. L., F. O’Flaherty, and P. F. Fox. 2006. Indigenous proteolytic
enzymes in milk: A brief overview of the present state of knowl-
edge. Int. Dairy J. 16:563–572.
Kerr, J. F. R., A. H. Wyllie, and A. R. Currie. 1972. Apoptosis: A
basic biological phenomenon with wide-ranging implications in
tissue kinetics. Br. J. Cancer 26:239–257.
Kitchen, B. J., G. Middleton, I. G. Durward, R. J. Andrews, and M.
C. Salmon. 1980. Mastitis diagnostic tests to estimate mammary
gland epithelial cell damage. J. Dairy Sci. 63:978–983.
Kornalijnslijper, J. E., A. J. J. M. Daemen, T. van Werven, T. A.
Niewold, V. P. M. G. Rutten, and E. N. Noordhuizen-Stassen.
2004. Bacterial growth during the early phase of infection deter-
mines the severity of experimental Escherichia coli mastitis in
dairy cows. Vet. Microbiol. 10:177–186.
Kuroishi, T., K. Komine, K. Asai, J. Kobayashi, K. Watanabe, T.
Yamaguchi, S. Kamata, and K. Kumagai. 2003. Inflammatory
responses of bovine polymorphonuclear neutrophils induced by
staphylococcal enterotoxin C via stimulation of mononuclear
cells. Clin. Diagn. Lab. Immunol. 10:1011–1018.
Lahteenmaki, K., P. Kuusela, and T. K. Korhonen. 2001. Bacterial
plasminogen activators and receptors. FEMS Microbiol. Rev.
25:531–552.
Lauzon, K., X. Zhao, A. Bouetard, L. Delbecchi, B. Paquette, and
P. Lacasse. 2005. Antioxidants to prevent bovine neutrophil-
induced mammary epithelial cell damage. J. Dairy Sci.
88:4295–4303.
Lauzon, K., X. Zhao, and P. Lacasse. 2006. Deferoxamine reduces
tissue damage during endotoxin-induced mastitis in dairy cows.
J. Dairy Sci. 89:3846–3857.
Ledbetter, T. K., M. J. Paape, and L. W. Douglas. 2001. Cytotoxic
effects of peroxynitrite, polymorphonuclear neutrophils, free
radical scavengers, inhibitors of myeloperoxidase, and inhibitors
of nitric oxide synthase on bovine mammary secretory epithelial
cells. Am. J. Vet. Res. 62:286–293.
Lee, J.-W., D. D. Bannerman, M. J. Paape, M.-K. Huang, and X.
Zhao. 2006. Characterization of cytokine expression in milk so-
matic cells during intramammary infections with Escherichia
 Tissue damage and mastitis
coli or Staphylococcus aureus by real-time PCR. Vet. Res.
37:219–229.
Lee, J., and X. Zhao. 2000. Recombinant human interleukin-8, but
not human interleukin-1β, induces bovine neutrophil migration
in an in vitro co-culture system. Cell Biol. Int. 24:889–895.
Leitner, G., O. Krifucks, U. Merin, Y. Lavi, and N. Silanikove. 2006.
Interactions between bacteria type, proteolysis of casein and
physico-chemical properties of bovine milk. Int. Dairy J.
16:648–654.
Leitner, G., E. Shoshani, O. Krifucks, M. Chaffer, and A. Saran. 2000.
Milk leucocyte population patterns in bovine udder infection of
different etiology. J. Vet. Med. B. 47:581–589.
Le Roux, Y., F. Laurent, and F. Moussaoui. 2003. Polymorphonuclear
proteolytic activity and milk composition change. Vet. Res.
34:629–645.
Li, X., X. Zhao, and S. Ma. 1999. Secretion of 92 kDa gelatinase
(MMP-9) by bovine neutrophils. Vet. Immunol. Immunopathol.
67:247–258.
Lin, Y., L. Xia, J. D. Turner, and X. Zhao. 1995. Morphological obser-
vation of neutrophil diapedesis across bovine mammary gland
epithelium in vitro. Am. J. Vet. Res. 56:203–207.
Long, E., A. V. Capuco, D. L. Wood, T. Sonstegard, G. Tomita, M. J.
Paape, and X. Zhao. 2001. Escherichia coli induces apoptosis and
proliferation of mammary cells. Cell Death Differ. 8:808–816.
Matsunaga, T., S. Kamata, N. Kakiichi, and K. Uchida. 1993. Charac-
teristics of Staphylococcus aureus isolated from peracute, acute
and chronic bovine mastitis. J. Vet. Med. Sci. 55:297–300.
Mebmer, U. K., V. A. Briner, and J. Pfeilschifter. 1999. Tumor necrosis
factor-α and lipopolysaccharide induce apoptotic cell death in
bovine glomerular endothelial cells. Kidney Int. 55:2322–2337.
Medan, D., L. Wang, X. Yang, S. Dokka, V. Castranova, and Y. Rojana-
sakul. 2002. Induction of neutrophil apoptosis and secondary
necrosis during endotoxin-induced pulmonary inflammation in
mice. J. Cell. Physiol. 191:320–326.
Mehrzad, J., C. Desrosiers, K. Lauzon, G. Robitaille, X. Zhao, and P.
Lacasse. 2005. Proteases involved in mammary tissue damage
during endotoxin-induced mastitis in dairy cows. J. Dairy Sci.
88:211–222.
Moir, E., N. A. Booth, B. Bennett, and L. A. Robbie. 2001. Polymorpho-
nuclear leukocytes mediate endogenous thrombus lysis via a u-
PA-dependent mechanism. Br. J. Haematol. 113:72–80.
Monks, J., F. J. Geske, L. Lehman, and V. A. Fadok. 2002. Do inflam-
matory cells participate in mammary gland involution? J. Mam-
mary Gland Biol. Neoplasia 7:163–176.
Moussaoui, F., F. Vangroenweghe, K. Haddadi, Y. Le Roux, F.
Laurent, L. Duchateau, and C. Burvenich. 2004. Proteolysis in
milk during experimental Escherichia coli mastitis. J. Dairy Sci.
87:2923–2931.
Nickerson, S. C., and C. W. Heald. 1981. Histopathologic response
of the bovine mammary gland to experimentally induced Staphy-
lococcus aureus infection. Am. J. Vet. Res. 42:1351–1355.
Ohta, S., K. Niiya, N. Sakuragawa, and H. Fuse. 2000. Induction of
urokinase-type plasminogen activator by lipopolysaccharide in
PC-3 human prostatic cancer cells. Thromb. Res. 97:343–347.
Oliver, S. P., and L. F. Calvinho. 1995. Influence of inflammation on
mammary gland metabolism and milk composition. J. Anim.
Sci. 73(Suppl. 2):18–33.
Owen, C. A., and E. J. Campbell. 1999. The cell biology of leukocyte
mediated proteolysis. J. Leukoc. Biol. 65:137–150.
Paape, M., D. Bannerman, X. Zhao, and J. Lee. 2003. The bovine
neutrophil: Structure and function in blood and milk. Vet. Res.
34:597–627.
Paape, M., J. Mehrzad, X. Zhao, J. Detilleux, and C. Burvenich. 2002.
Defense of the bovine mammary gland by polymorphonuclear
65
neutrophil leukocytes. J. Mammary Gland Biol. Neoplasia
7:109–121.
Pitkala, A., M. Haveri, S. Pyorala, V. Myllys, and T. Honkanen-
Buzalski. 2004. Bovine mastitis in Finland 2001—Prevalence,
distribution of bacteria, and antimicrobial resistance. J. Dairy
Sci. 87:2433–2441.
Politis, I. 1996. Plasminogen activator system: Implications for mam-
mary cell growth and involution. J. Dairy Sci. 79:1097–1107.
Prin-Mathieu, C., Y. Le Roux, G. C. Faure, F. Laurent, M. C. Béné,
and F. Moussaoui. 2002. Enzymatic activities of bovine periph-
eral blood leukocytes and milk polymorphonuclear neutrophils
during intramammary inflammation caused by lipopolysaccha-
ride. Clin. Diagn. Lab. Immunol. 9:812–817.
Raulo, S. M., T. Sorsa, T. Tervahartiala, T. Latvanen, E. Pirila, J.
Hirvonen, and P. Maisi. 2002. Increase in milk metalloprotei-
nase activity and vascular permeability in bovine endotoxin-
induced and naturally occurring Escherichia coli mastitis. Vet.
Immunol. Immunopathol. 85:137–145.
Riollet, C., P. Rainard, and B. Poutrel. 2000a. Cells and cytokines in
inflammatory secretions of bovine mammary gland. Adv. Exp.
Med. Biol. 480:247–258.
Riollet, C., P. Rainard, and B. Poutrel. 2000b. Differential induction
of complement fragment C5a and inflammatory cytokines during
intramammary infections with Escherichia coli and Staphylococ-
cus aureus. Clin. Diagn. Lab. Immunol. 7:161–167.
Rudolph-Owen, L. A., and L. M. Matrisian. 1998. Matrix metallopro-
teinases in remodeling of the normal and neoplastic mammary
gland. J. Mam. Gland Biol. Neoplasia 3:177–189.
Sheffield, L. G. 1997. Mastitis increases growth factor messenger
ribonucleic acid in bovine mammary glands. J. Dairy Sci.
80:2020–2024.
Sordillo, L. M., and S. C. Nickerson. 1988. Morphologic changes in
the bovine mammary gland during involution and lactogenesis.
Am. J. Vet. Res. 49:1112–1120.
Sordillo, L. M., and K. L. Streicher. 2002. Mammary gland immunity
and mastitis susceptibility. J. Mammary Gland Biol. Neoplasia
7:135–146.
Strange, R., F. Li, S. Saurer, A. Burkhardt, and R. R. Friis. 1992.
Apoptotic cell death and tissue remodeling during mouse mam-
mary gland involution. Development 115:49–58.
Takahashi, H., T. Komatsu, K. Hodate, R. Horino, and Y. Yokomizo.
2005. Effect of intramammary injection of RbIL-8 on milk levels
of somatic cell count, chemiluminescence activity and shedding
patterns of total bacteria and S. aureus in Holstein cows with
naturally Infected-subclinical mastitis. J. Vet. Med. B 52:32–37.
Trinidad, P., S. C. Nickerson, and R. W. Adkinson. 1990. Histopathol-
ogy of staphylococcal mastitis in unbred dairy heifers. J. Dairy
Sci. 73:639–647.
Ward, P. A., J. S. Warren, and K. J. Johnson. 1988. Oxygen radicals,
inflammation, and tissue injury. Free Radical Bio. Med.
5:403–408.
Weinrauch, Y., and A. Zychlinsky. 1999. The induction of apoptosis
by bacterial pathogens. Annu. Rev. Microbiol. 53:155–187.
Wesson, C. A., J. Deringer, L. E. Liou, K. W. Bayles, G. A. Bohach,
and W. R. Trumble. 2000. Apoptosis induced by Staphylococcus
aureus in epithelial cells utilizes a mechanism involving Cas-
pases 8 and 3. Infect. Immun. 68:2998–3001.
Wesson, C. A., L. E. Liou, K. M. Todd, G. A. Bohach, W. R. Trumble,
and K. Bayles. 1998. Staphylococcus aureus Agr and Sar global
regulators influence internalization and induction of apoptosis.
Infect. Immun. 66:5238–5243.
Wilson, D. J., R. N. Gonzales, and H. H. Das. 1997. Bovine mastitis
pathogens in New York and Pennsylvania: Prevalence and ef-
fects on somatic cell count and milk production. J. Dairy Sci.
80:2592–2598.