The Mammary Microenvironment in Mastitis in Humans, Dairy Ruminants, Rabbits and Rodents: A One Health Focus
Journal of Mammary Gland Biology and Neoplasia
The Mammary Microenvironment in Mastitis in Humans, Dairy Ruminants, Rabbits and Rodents: A One Health Focus
Katherine Hughes 0 1
Christine J. Watson 0 1
0 Department of Pathology, University of Cambridge , Tennis Court Road, Cambridge CB2 1QP , UK
1 Department of Veterinary Medicine, University of Cambridge , Madingley Road, Cambridge CB3 0ES , UK
The One Health concept promotes integrated evaluation of human, animal, and environmental health questions to expedite advances benefiting all species. A recognition of the multi-species impact of mastitis as a painful condition with welfare implications leads us to suggest that mastitis is an ideal target for a One Health approach. In this review, we will evaluate the role of the mammary microenvironment in mastitis in humans, ruminants and rabbits, where appropriate also drawing on studies utilising laboratory animal models. We will examine subclinical mastitis, clinical lactational mastitis, and involution-associated, or dry period, mastitis, highlighting important anatomical and immunological species differences. We will synthesise knowledge gained across different species, comparing and contrasting disease presentation. Subclinical mastitis (SCM) is characterised by elevated Na/K ratio, and increased milk IL-8 concentrations. SCM affecting the breastfeeding mother may result in modulation of infant mucosal immune system development, whilst in ruminants notable milk production losses may ensue. In the case of clinical lactational mastitis, we will focus on mastitis caused by Staphylococcus aureus and Escherichia coli. Understanding of the pathogenesis of involution-associated mastitis requires characterization of the structural and molecular changes occurring during involution and we will review these changes across species. We speculate that milk accumulation may act as a nidus for infection, and that the involution 'wound healing phenotype' may render the tissue susceptible to bacterial infection. We will discuss the impact of concurrent pregnancy and a 'parallel pregnancy and involution signature' during bovine mammary involution.
Mammary gland; Mastitis; Microenvironment; One Health; Rabbit; Ruminant; Sheep
CAEV Caprine arthritis and encephalitis virus
CLCA Chloride channel regulators, calcium activated
E Embryonic day
FR Fürstenberg's rosette
HIV Human immunodeficiency virus
LIF Leukemia inhibitory factor
LTA Lipoteichoic acid
MMP Matrix metalloproteinase
MVV Maedi-visna virus
In this review, we will evaluate the role of the mammary
microenvironment in mastitis in humans, ruminants and
rabbits, where appropriate also drawing on studies utilising
laboratory animal models. We will examine subclinical mastitis,
clinical lactational mastitis, and involution-associated, or dry
period, mastitis, which is more common in ruminants than in
humans. We will synthesise knowledge gained across
different species, comparing and contrasting disease presentation
between humans and other species. For clarity and precision,
the term ‘breast’ will be used solely to refer to the human
mammary gland in the ensuing discussion.
Breast pain is one of the two most common problems faced
by breastfeeding women [
]. Mastitis affects up to 33% of
lactating mothers and is a major cause of precocious weaning [
], with the concomitant short- and long-term health
consequences for the child [
]. In addition, it has recently been
suggested that subclinical mastitis may influence the infant’s
mucosal immune system [
]. Similarly, in dairy cows and sheep, both
acute clinical disease and chronic subclinical mastitis cause
significant pain [
] and thus present a major welfare problem.
Mastitis represents around a third of the direct costs of all
common dairy diseases  and has public health repercussions,
particularly associated with the increased use of antimicrobials for
disease treatment. Mastitis is also a major health concern in
breeding does on rabbit farms [
]. Indeed, mastitis has been
recorded amongst the most common reasons for culling does in
Spanish commercial rabbitries  with mastitis the reason for
euthanasia of 33% of the females culled in one study [
]. In a
separate study of rabbit farms in Spain and Portugal, the
prevalence of mastitis was 4% in lactating does [
]. In addition,
mastitis may occur in pet female rabbits that are lactating or that
develop pseudopregnancy. Mammary gland trauma or poor
hygiene may be predisposing factors in such cases [
One Health is a term embodying the notion that there is a
complex interplay between human and veterinary medicine
and the environment, all of which impact human and animal
]. This concept can be applied to specific problems
in which human and animal health clearly intersect, such as
zoonotic diseases, or the responsible usage of antimicrobials
], but the One Health focus of biomedical research also
extends beyond such specific issues to promote an integrated
evaluation of human, animal, and environmental health
questions in a manner conducive to expediting conceptual
advances and meaningful solutions benefiting all species [
One Health thus offers recognition that both human and
veterinary medicine can Bcontribute to the development of each
] and, amongst many other topics, encompasses
molecular and microbiology as they relate to comparative
and translational medicine [
]. Given the multi-species
impact of mastitis as a painful condition potentially influencing
the welfare of both the mother and offspring, together with its
wider economic and public health implications, we suggest
that mastitis is an ideal target for a One Health approach.
The mammary microenvironment comprises luminal and
basal epithelial cells, stromal components including
fibroblasts, endothelial cells and stromal matrix, and the immune
cell compartment. These mammary constituents engage in a
complex and interweaving network of interactions, with
physical connectivity, and paracrine and hormonal influences,
orchestrating the interplay. Pathogen biology is frequently at the
forefront of mastitis studies, but it is evident that the response
of the mammary epithelium and the microenvironment to the
pathogen is also critical (Fig. 1), and elements of the
microenvironment may provide novel therapeutic targets [
Comparative Mammary Gland Anatomy and Development
One challenge associated with consideration of mastitis from a
One Health perspective revolves around an appreciation of
species differences in anatomy which may impact the
pathogenesis of mastitis. For example the sinuses present in the
ruminant mammary gland have a particular role in storage of
milk, and may provide a medium for bacterial growth, as well
as being a site where immune cells transit into the milk. Thus,
the following section will consider comparative mammary
gland anatomy and development, with twofold aims to both
emphasise structures that may have a role in mastitis
pathogenesis and to highlight differences between species.
The anatomy of the mammary gland has been studied for
] and species variations have been well-documented
(Table 1). In addition to the externally obvious variations in the
number of mammae, the number of galactophorous, or lactiferous,
ducts also varies, and this impacts the number of divisions per
mamma. Terminology in this field is variable, with some authors
preferring the term ‘gland’ to describe these divisions [
others prefer the terms ‘lobes’ [
], ‘sectors’ [
] or ‘ductal
]. We will use the term ‘ductal system’ to refer to this
division in the following discussion. Bovine, ovine, caprine and
murine mammae have one ductal system per mamma [
rabbit has approximately six or seven [
] (authors’ submitted
manuscript) and the human breast has 4–18 [
] (Table 1).
The important anatomical difference in the number of ductal
systems per mamma arises during development. At
approximately embryonic day (E) 15.5 in the female mouse embryo,
the mammary primordium transitions to the sprout stage and
the distal aspect of the bulb elongates to penetrate the deeper
mesenchyme, termed the secondary mammary mesenchyme,
(synonym: fat pad precursor mesenchyme). Subsequent
branching morphogenesis of the sprout produces a rudimentary
branched ductal system [
]. Similarly, in the rabbit, between
E17 and E23, the bulb’s spherical aspect becomes larger and
elongates to enter a deeper zone of adipose-rich mesenchyme.
However, by contrast to the mouse, at E26 in the rabbit, the bud
commences division, with each resulting sprout giving rise to a
primary milk canal that subsequently undergoes branching
morphogenesis. Hence the rabbit (and human) mamma exhibit
multiple mammary trees, each with a primary milk canal
dividing the mamma into different ductal systems [
] (Fig. 2).
Comprehensive descriptions of mammary gland
development in the human [
], mouse [
], ruminant 
and rabbit [
] already exist and this subject will only be
discussed further in this review with respect to aspects of
postnatal development, in particular post-lactational
regression, which are relevant to the pathogenesis of mastitis arising
in association with the ruminant dry period. In dairy cows, the
dry period, particularly the time immediately following the
end of the prior lactation and the time immediately preceding
Fig. 1 The mammary microenvironment in mastitis in a third
lactation Holstein Friesian cow, 46 dL. (a) Multifocal mammary
alveoli are engorged with numerous predominantly degenerate
neutrophils (*). Haematoxylin and eosin stain; scale bar: 300 μm. (b)
Severely affected alveoli with myriad neutrophils (*) exhibit partial loss
of the luminal epithelial lining (arrows) although the partial remnants of
the mammary epithelial lining remain (double headed arrow).
Haematoxylin and eosin stain; scale bar: 50 μm. (c) Scattered
aggregates of lymphocytes expressing CD3 (arrowhead) are present
multifocally. Immunohistochemical staining for CD3 with haematoxylin
counterstain; scale bar: 100 μm. (d) Rarer individual lymphocytes
expressing CD20 (arrowhead) are present between mammary alveoli.
Immunohistochemical staining for CD20 with haematoxylin
counterstain; scale bar: 100 μm. dL: days lactation
parturition, represents a phase of the mammary cycle when
new intramammary infections may be acquired [
In ruminants, the larger ducts from each mamma open into
the gland cistern, which in turn communicates with the teat
cistern that, itself, is connected with the exterior via the teat
] (Fig. 3). Early dissections describe the ductal
anatomy of the lactating breast . More recent studies have
included three dimensional reconstruction from serial
histological sections allowing the appreciation of ductal anatomy in
three dimensions [
]. Magnetic resonance imaging has also
been employed to delineate changes in breast morphology
between lactation and weaning [
]. Although earlier work has
depicted lactiferous sinuses within the breast [
], an ultrasound
study of 21 lactating women demonstrated a frequent increase
in duct diameter at multiple branch points, but an absence of
lactiferous sinuses under the areola, leading the authors of that
study to conclude that in the human breast, ducts act as a milk
conduit rather than a storage sinus. Importantly, these authors
also noted that the ducts were easily compressed [
Lactiferous sinuses have not been described in the mammary
Fig. 2 Sub-gross anatomy and
histology of the rabbit
mammary gland. (a) Sub-gross
histological section (sagittal
plane) through the teat and
mammary tissue of a wild rabbit,
Oryctolagus cuniculus, during
late pregnancy, estimated 27 dG.
Multiple ducts (*) are apparent
and exhibit dilatations suggestive
of sinusoidal structures (S). (b)
Transverse section of a rabbit teat
canal, < 1 mm from the teat
orifice, demonstrating the
keratinized stratified squamous
epithelium. (c) Transverse section
of a mammary duct
demonstrating the bilaminar
epithelial lining (double headed
arrow). (d) Mammary alveoli
formed by a luminal layer of
mammary epithelial cells and an
underlying layer of
and eosin stain; dG: days
gland of the rabbit [
] and it has been suggested that milk is
stored in the secretory alveoli [
] although we observe
sinuslike dilatations of the milk ducts in the mammary gland of
pregnant and lactating rabbits (Fig. 2) (submitted manuscript).
The mammary stroma also varies between species. In
rodents, the mammary fat pad is predominantly composed of
adipocytes. In the lactating breast, the stroma is also adipose-rich
], whilst in the ruminant, the stroma comprises adipose
dissected by trabeculae of collagen [
]. Interestingly, in the
developing bovine mammary gland, authors recognise ‘far
stromal’ regions comprising a combination of adipose and fibrous
tissue, and more dense ‘near stromal’ tissue regions which are
arranged within a 100 to 150 μm radius of mammary epithelial
structures . We have observed similar ‘far stromal’ and ‘near
stromal’ zones in the mammary gland of ewe lambs (Fig. 4).
Comparative Mammary Gland Immunology
It is not the purpose of this review to provide a comprehensive
account of immunology of the mammary gland, and, indeed,
other authors have provided detailed insights into this subject
in humans and mice [
] and dairy ruminants [
However, it is pertinent to mention several aspects of
comparative mammary gland immunology which are particularly
relevant to the study of mastitis from a One Health perspective.
Teat Sphincter, Teat Canal, Fürstenberg’s Rosette,
and Teat Cistern
Several elements of the ruminant mammary immune defences
derive from the particular anatomy of the udder as described
]. The teat sphincter is an important anatomical
structure maintaining closure of the teat canal [
Recently, cells situated near smooth muscle cells and
exhibiting cytoplasmic processes, and dual positivity for
CD117 and vimentin, have been identified and suggested to
represent bovine telocytes. It has been postulated that these
cells may have a role in regulating the contractility of the
sphincter and thus may act as a component of the innate
immune system of the ruminant mammary gland .
Located at the proximal end of the bovine teat canal (Fig.
3), the Fürstenberg’s rosette (FR) is a small region between the
streak canal and the teat cistern which has 4–18 folds and is 2–
11 mm in width, [
]. It is not clear whether the FR solely
forms an integral component of the physical barrier of the teat
or whether it has a more specific immunological role.
However, at the mRNA expression level, the FR exhibits
higher constitutive expression of S100 proteins (A8, A9,
A12) than the teat cistern, perhaps implying a distinct
immunological function [
]. Intriguingly, the FR is described as
having a protective leukocyte population, and the presence of
intraepithelial leukocytes suggests that some of these immune
cells leave the teat wall and enter the cistern [
] (Fig. 3).
In a large histological study of ovine teats from clinically
healthy animals, lymphocytes were the predominant immune
cell type in the teat cistern, whereas in the teat duct, similar
numbers of lymphocytes and neutrophils were observed.
Subepithelial lymphoid nodules, most frequently at the border
between teat duct and teat cistern, were detected in 49% of the
samples examined, and were significantly associated with the
presence of bacteria [
]. Thus, in sheep, it also seems likely
that the presence of subepithelial lymphoid tissue at the border
between teat duct and teat cistern is an important component of
mammary defence but to fully elucidate the role of these
aggregates it is critical to distinguish between pre-existing lymphoid
accumulations and those induced by bacterial invasion [
This is an example of an area in which a One Health approach
may offer useful insights for future research and where the
relevance of findings derived from one species is yet to be
tested in other species. For example, we observe lymphocytes
at the junction between the teat canal and the mammary duct in
rabbits (authors’ unpublished data) but, as yet, the significance
of these groupings of lymphocytes remains undefined.
histological inset: Sagittal section through the teat canal (TC) – distal
teat cistern (diamond) junction, Fürstenberg’s rosette (*) of a Holstein
Friesian dairy cow 45 dI. Groupings of small to moderate numbers of
lymphocytes (L) are present multifocally. Haematoxylin and eosin stain;
dI: days involution, with concurrent pregnancy until abortion at
The Mammary Epithelial Cell as A Component Of
The Mammary Immune System
It is well-established that mammary epithelial cells themselves
are important players in the mammary immune
microenvironment. We have previously demonstrated that, during murine
post-lactational regression, mammary epithelial cells exhibit
Fig. 4 Near- and far- stromal regions of the immature ovine
mammary gland. Histolological section from the mammary gland of a
nulliparous 18-month-old ewe lamb. Near-stromal regions (N) are within
approximately 75 μm of mammary epithelial structures. Far-stromal
regions (F) comprise both collagen rich connective tissue and adipose.
Haematoxylin and eosin stain; scale bar: 50 μm
dramatic, Stat3-dependent, up-regulation of CD14, an innate
immune component [
]. In addition, cultured bovine and
caprine mammary epithelial cells have been shown to express
TLR2 and TLR4 mRNA, and responses of mammary epithelial
cells to stimulation with bacterial cell wall components, or other
agents with the potential to cause mastitis, such as Prototheca
spp., result in elaboration of a plethora of cytokines and other
inflammatory mediators [
]. These findings suggest that
mammary epithelial cells, across species, have a critical role to
play in the mammary immune microenvironment although
clearly much of the current evidence for this assertion is derived
from studies using rodent mammary epithelial cells in vitro, and
from in vivo studies of rodent models.
Mammary epithelial cells are also recognised to have
phagocytic capability, perhaps best illustrated by work demonstrating
the role of mammary epithelial cells in the process of
efferocytosis. Efferocytosis comprises the phagocytic removal
of superfluous or damaged cells by either neighbouring tissue
cells or professional phagocytes [
] and is particularly
important during mammary gland involution, the process by which
the glandular architecture is remodelled at the cessation of
lactation. It has been demonstrated that murine mammary
epithelial cells are important effectors of efferocytosis during
involution, and that this process is dependent on the autophagy related
proteins Becn1 and ATG7 [
] and the receptor tyrosine kinase
]. Interestingly, the study of the mammary gland of
sheep has also yielded morphological evidence of epithelial cell
efferocytosis during mammary gland involution [
Mammary epithelial cells also use phagocytosis to take up large
milk fat globules accumulating in the mammary alveolar lumen
during early involution [
], further underlining the ability of
the glandular epithelial cells to acquire a phagocytic phenotype.
Soluble Components of Mammary Gland Immunity
There are a number of soluble proteins that constitute important
components of mammary gland immunity, including lactoferrin,
transferrin, the lactoperoxidase and myeloperoxidase systems,
complement proteins, and lysozyme [
]. However, drawing
comparisons across species necessitates an awareness of species
variations in the synthesis of such components, and the
concentrations of these components in milk. For example, lysozyme is
a bactericidal protein present in milk which catalyses the
hydrolysis of peptidoglycan residues in bacterial cell walls. Lysozyme
appears to be an important immune component of human milk
but is present at a much lower concentration in bovine milk [
The term subclinical mastitis (SCM) is used to indicate
inflammation of the breast or mamma which does not result in
clinically detectable symptoms. A notable condition in both humans
and milk production animals, SCM is associated with decreased
milk quality and yield in dairy ruminants [
] and presumably a
similar phenomenon in humans, where it has been associated
with reduced infant weight gain [
]. Importantly, it is
increasingly recognised that breast milk consumption may influence
the immunological development of babies [
] and that
SCM may therefore impact this development . In addition,
SCM has particularly significant potential sequelae for infants
breast fed by women with lentiviral infections, and this topic
will be discussed further at the end of this section.
In humans, diagnosis of SCM is reached when there is an
elevated milk sodium/potassium (Na/K) ratio above 1.0, and
an increased concentration of interleukin-8 (IL-8), in the
absence of clinical signs [
]. In dairy ruminants, milk somatic
cell count (SCC) or microbial culture-based methods are
mainstays of diagnosis [
The study of SCM is particularly relevant to a One Health
approach as human SCM is relatively poorly characterised,
whereas bovine SCM, in particular, is a well-established research
focus due to the high proportion of mastitis-associated losses
attributed to SCM. Therefore it is possible, that testing insights
from bovine SCM for their applicability to human SCM may be
a fruitful avenue of future research in the One Health field.
However, any such approach would require an outlook mindful
of species differences as discussed elsewhere in this review.
Changes in Milk Na/K Ratio During SCM
Fluctuations in the mammary microenvironment in human
SCM are relatively poorly understood and alterations in milk
components provide the main ‘window’ into understanding
the changes which may be occurring in the breast at this time.
As already mentioned, the milk Na/K ratio is perturbed during
an episode of SCM, providing a diagnostic tool. To
understand this phenomenon, it is necessary to consider changes
occurring at the level of both the whole mammary gland,
and individual mammary epithelial cells.
Once lactation has been established (at approximately day
four post partum) breast milk Na/K ratio is primarily affected by
extent of milk extraction from the gland. When demand for
milk is reduced, which could be due to factors including breast
feeding technique or supplementary feeding, the gland becomes
distended. This potentially exacerbates reduced emptying, as
the infant’s ability to suckle from an engorged breast may be
compromised or the breast may be more prone to injury [
When considering breast engorgement as a factor in the
development of subclinical mastitis, it is important to note that, as
already described, in humans, milk ducts act as a milk channel
rather than a storage sinus, and are readily compressible [
Furthermore, the processes described above can also contribute
to a ‘vicious cycle’ where mastitis itself can contribute to
compromised milk removal and breast engorgement [
The secretory activation phase of differentiation
(formerly termed lactogenesis stage 2), prompted by
progesterone withdrawal, commences at the termination of
pregnancy in rodents and ruminants and shortly after
parturition in humans, when the placenta is delivered [
Importantly, secretory activation heralds the closure of
tight junctions (synonym: zona occludins) between
mammary epithelial cells . However, if demand for milk
declines, the mammary acini are progressively distended,
the tight junctions between epithelial cells open and
sodium influx into the milk increases. Leaky tight junctions,
the consequent unrestricted movement of components of
the interstitial space into the milk, and thus an elevated
milk Na/K ratio, are important features of subclinical
]. Although the gland will tend to down-regulate
milk production to match demand, milk stasis may
potentially lead to infection [
]. Buffaloes with SCM diagnosed
on the basis of bacteriology and SCC also exhibit elevated
milk Na and decreased K [
], further indicating that a
perturbed Na/K ratio may be a feature of SCM irrespective
In parallel with an increased Na/K ratio, milk
concentrations of IL-8 increase in human SCM [
Interestingly, using quantitative real time polymerase chain
reaction, expression of the IL-8 receptor has also been
demonstrated to be significantly higher in milk somatic
cells from crossbred dairy cows diagnosed with SCM,
detected on the basis of the California Mastitis Test, SCC,
and electrical conductivity test, when compared to healthy
]. A recent study has demonstrated that human
SCM, as defined by an Na/K ratio in excess of 1, is
associated with higher levels of a panel of inflammatory
mediators and markers, again including IL-8, but also including
β2 microgobulin, PS100A9, TNF-α, IL-6, IL-17,
RANTES, IL-2R, IL-12p40/70, IFN-α, IFN-γ, CXCL-9,
IP-10, MIP-1α, MIP-1β, LPS binding protein,
αdefensins, and antileukoproteinase 1 [
]. Again, evidence
of a similar inflammatory profile can be inferred in dairy
ruminants, where higher levels of TNF-α and IL-12 have
been observed in ewes with SCM [
SCM has been implicated as a potential risk factor
which increases the likelihood of mother-to-child Human
Immunodeficiency Virus (HIV) transmission via breast
]. Staphylococcus aureus is a frequently
implicated pathogen in HIV-infected women in Malawi [
The small ruminant lentiviruses (SRLVs) caprine arthritis
and encephalitis virus (CAEV), maedi-visna virus (MVV),
and ovine progressive pneumonia virus (OPPV) may also
be shed in colostrum and milk . Whilst it is
wellestablished that SRLVs may cause mastitis, to the authors’
knowledge, it is not known specifically whether the
presence of SCM increases the likelihood of SRLV vertical
Clinical Lactational Mastitis
The normal bacterial content of human breast milk is
increasingly recognised as critical to infant immune development
] and thus lactational mastitis presents particular
challenges in human subjects where inappropriate antibiotic
therapy for patients with lactational mastitis is likely to have
profound effects on the microbiome . Interestingly, in
humans, the microbiome of mastitic milk has been
demonstrated to have a reduced bacterial diversity [
veterinary medicine, much of the focus of mastitis research has
centred upon the pathogenic organisms involved, which has
led to a better appreciation of the epidemiology and
pathogenesis of mastitis in the context of different pathogens. In 2014 a
new paradigm for mastitis was proposed, emphasising the
importance of host inflammatory mediators as drivers of
disease . We suggest that both approaches are relevant to the
study of mastitis with a One Health focus and our discussion
of lactational mastitis will focus on the microenvironmental
response to two key mastitis pathogens. It is pertinent to
emphasise here that taking a ‘pathogen approach’ to
consideration of microenvironmental changes is not advocating use
of antibiotic therapy in cases of human lactational mastitis.
In one human study, Staphylococcus aureus was the most
frequently isolated pathogenic bacterium cultured from the
breast milk of women with lactational mastitis [
the results of such studies must be interpreted with caution as
other authors have identified the presence of potentially
pathogenic bacteria in the breast milk of healthy women [
Bovine and ovine mastitis arising due to S. aureus infection
may vary in severity, with a highly pathogenic, necrotizing, or
gangrenous, form the most severe manifestation. Many cows
also exhibit chronic mastitis or subclinical mastitis as a result
of S. aureus infection [
Rabbits are also susceptible to S. aureus mastitis. This may
present as a spectrum of lesions ranging from acute and
necrotizing (gangrenous) forms [
], to more commonly chronic,
suppurative, lesions [
9, 11, 13
] with variable levels of
]. In rabbits, the primary granulocytic leukocyte is
frequently termed the heterophil rather than the neutrophil
. The diameter of a heterophil is 7–10 μm, the nucleus
frequently exhibits multiple lobes and the cytoplasm exhibits
eosinophilic granules [
]. Following lymphocytes,
heterophils are the second most frequently encountered leucocyte
in the rabbit, and comprise 20–75% of white blood cells
]. Thus, purulent lesions in this species are microscopically
characterised by accumulations of viable and degenerate
]. Lesion cellularity in staphylococcal mastitis in
rabbits may reflect mastitis chronicity, with greater numbers
of T lymphocytes, macrophages and plasma cells in immature
In chronic S. aureus infections in bovine tissue, more
IgG1and IgG2- secreting leukocytes were detected at the FR when
compared to uninfected quarters [
]. Experimental bovine
udder challenge with S. aureus isolate 1027 (subclinical
mastitis) has shown that there is an immune response by one hour
after pathogen challenge, with up-regulation of expression of
CCL20, CXCL8, TNF and IL-6 in the teat cistern [
]. In a
longer time course udder inoculation model using another
s u b c l i n i c a l m a s t i t i s - c a u s i n g s t r a i n o f S . a u re u s
(NCTC13047), IL-6, IL-17A, IL-8, and IL-10 were induced
in the alveoli, ducts, gland cistern and teat canal of the bovine
mammary gland. Expression of the acute phase proteins
serum amyloid A3 and haptoglobin was induced, together with
antimicrobial peptides [
]. Other investigators have
demonstrated that intramammary challenge with lipoteichoic acid
(LTA) from S. aureus will also increase mRNA expression
of various cytokines including TNF-α, IL-1β, IL-8 and
RANTES, and decreased lactoferrrin expression [
]. It has
been suggested that the transcriptional changes documented in
the bovine udder following challenge with S. aureus most
likely echo the dual contributions of mammary epithelial cell
activation and immune cell influx to changing gene
expression profiles [
]. It has also been demonstrated that S. aureus
can modulate Rho GTPase regulated pathways. Using actin
fibres stained with fluorescent-labelled phalloidin, the same
investigators have shown that primary bovine mammary
epithelial cells cultured with S. aureus have an altered, more
filamentous, actin cytoskeleton, which may facilitate bacterial
Excitingly, a recent study has demonstrated that, in a
murine model, preconditioning the mammary gland with
inoculation of LTA or lipopolysaccharide (LPS) modulates the
innate immune response to a local S. aureus infection, and
reduces the subsequent bacterial burden. By depleting
macrophages in this model, the authors showed that this response
was partially independent of macrophage signalling and the
authors also implicated lipocalin 2 and chitinase 3-like 1 as
potential modulators of the innate immune response [
Fibroblasts have an important role in chronic S. aureus
mastitis as they facilitate collagen deposition and may mediate
extensive glandular fibrosis. It has been suggested that
transforming growth factor beta-1 may enhance S. aureus
adhesion to, and invasion of, bovine mammary fibroblasts and
that this interaction may be reduced using ERK inhibitors [
In the cow, severe, necrotising, coliform mastitis may result
from infection, with cows frequently also presented with
concurrent endotoxaemia. Milder disease forms have also been
]. Experimental intramammary administration of
bacterial endotoxin results in the sloughing of large numbers
of epithelial cells into milk [
]. In the same series of
experimental bovine udder challenges as already described for
S. aureus, challenge with E. coli resulted in enhanced
upregulation of expression of a wider panel of immune factors
than S. aureus, with increases seen in CCL20, CXCL8, TNF,
IL-6, IL-12b, IL-10, LAP and S100A9. However, the E. coli
isolate used was a clinical mastitis isolate (1303) whereas the
S. aureus strain was an isolate from a subclinical infection
]. Intramammary challenge with LPS also shows stronger
induction of expression of TNF-α, IL-1β, IL-8 and RANTES
than LTA challenge, and similar decreased lactoferrrin
expression. LPS challenge elicits increased concentrations of TNF-α
in milk [
A microarray study in which cows received experimental
intramammary administration of E. coli four to six weeks after
calving has demonstrated that, in total, 982 transcripts are
differentially expressed during the bovine host response to
E. coli mastitis [
], with a wide network of pathways
affected including complement and coagulation cascades,
JakSTAT signalling pathways and the toll-like receptor (TLR)
signalling pathway (reviewed by [
]). The importance of
TLR signalling is underlined by a study using TLR4 null
mutant mice in which induction of mastitis via introduction
of LPS resulted in initial higher abundance of neutrophils and
macrophages but a reduced lesion infiltration of the same
inflammatory cell types at 7 days post inoculation. Serum
concentrations of certain cytokines, including CXCL1, CCL2,
IL1β, and TNF-α, were also reduced compared to wild-type
]. However, following resolution of mastitis, milk
production capacity was reduced in wild-type mice
compared to those deficient in TLR4, raising the possibility that
mastitis-associated lactation insufficiency may be due in
part to TLR4-mediated inflammation, rather than bacterial
infection per se [
Involution- or Dry Period-Associated Mastitis
Mammary gland involution comprises the remodelling of the
gland at the end of the lactation period. In humans, mastitis
may potentially be associated with weaning particularly if the
process is insufficiently gradual and the breast becomes
engorged, with the pathogenesis potentially similar to that
described to account for the elevated Na/K ratio used as a
diagnostic modality in subclinical mastitis.
In ruminants, the start of involution, or the dry period, is
well-recognised as a time when there is an increased
likelihood of acquisition of new mammary infections, especially
in cows with high milk yields prior to drying off [
‘Summer mastitis’ is mastitis of dairy cows occurring
during the summer months and therefore, in traditional
systems, is associated with the dry period. This condition is
usually caused by mixed bacterial species including
Trueperella pyogenes and Streptococcus dysgalactiae.
The route of infection is thought to be the papillary ostium
and duct, and flies attracted to pre-existing teat lesions are
implicated in the pathogenesis of the disease. In traditional
systems, some of the affected animals at pasture would be
dry, whilst others might be immature heifers [
In dairy small ruminants, it is suggested that pre-existing
intramammary infections may recrudesce during the dry
period and thus result in clinical disease. It is also documented that
ewes/does are particularly susceptible to new infections [
In addition, in specific instances, a high incidence of mastitis
is rarely seen at drying off in association with specific
pathogens such as Pseudomonas aeruginosa or fungal agents [
Given the dramatic molecular and structural changes which
occur in the mammary gland during post-lactational
remodelling, we suggest that when considering episodes of dry
periodor involution-associated mastitis, it may be informative to
consider changes occurring in the mammary
microenvironment during involution and to speculate on how these changes
may influence susceptibility of the gland to mastitis-causing
pathogens at this time. We will adopt this approach in the
Species Differences in the Mammary
Microenvironment During Involution
Excitingly, a recent study used magnetic resonance imaging to
describe changes seen in the breast during the first year
postweaning, and the investigators noted that the gland returns to a
state similar to that observed pre-conception. Both breast area
and fibroglandular fraction decreased significantly between
women in the lactation and post weaning periods and
measurements for the latter group were comparable to those of a
premenopausal control group [
]. Such a striking degree of
glandular remodelling is the result of highly regulated,
interconnecting, networks of cellular signals which
orchestrate the involution process [
]. Set at the hub of this
molecular system is the transcription factor Signal Transducer
and Activation of Transcription 3 (STAT3) [
STAT3 activation is fundamental to the normal progression
of involution [
Mammary gland involution is considered to progress in
two distinct phases, which have been well-defined in the
mouse. The first reversible phase is proteinase independent
and is characterised by dramatic mammary epithelial cell
death co-ordinated by Stat3 [
]. Mice with a
mammary-specific conditional deletion of Stat3 exhibit a
p r o n o u n c e d r e t a r d a t i o n o f i n v o l u t i o n [
1 0 9 , 11 0
Unilateral teat sealing, in which mice have sealant
unilaterally applied to the inguinal mammary gland teat, such that
pups sucking the contralateral open gland provide a
continued systemic suckling stimulus, has demonstrated that local
factors, attributed to milk accumulation, are sufficient to
induce phosphorylation of Stat3 and consequent cell death
at the onset of involution [
]. Leukemia inhibitory factor
(LIF) exhibits a rapid increase in expression, which is
independent of systemic factors, and is accordingly observed
even in teat-sealed glands [
]. LIF deficient mice exhibit
delayed mammary regression and absence of Stat3
activation during involution [
], demonstrating that the initial
activator for Stat3 in the mammary gland is LIF, and that the
up-regulation of pStat3 at the onset of involution is
independent of the decrease in circulating lactogenic hormones
seen after weaning. However, those glands which are sealed
with the contralateral gland left open (thus maintaining
systemic hormone stimulation) do not progress to the second
phase of involution [
]. Thus exogenous administration
of hydrocortisone, or systemic factors such as endogenous
glucocorticoid release, can inhibit progression to the
second phase [
When progression of involution is unimpeded, the second
phase is accompanied by irreversible degradation of the
mammary basement membrane, coinciding with expression, by
fibroblasts and other mesenchymal components, of the matrix
metalloproteinases (MMPs) MMP2 (gelatinase A), and
MMP3 (stromelysin 1), the serine proteinase urokinase-type
plasminogen activator [
], and MMP9 [
During the early phase of involution in mice, there is
dramatic up-regulation of genes associated with the acute phase
response and innate immunity, including serum amyloid A3
]. Stat3 regulates expression of a subset of these genes,
including orosomucoids 1 and 2, secretory leukocyte protease
inhibitor, CD14 and leucine-rich α2-glycoprotein 1 [
The later stages of involution are characterised by the
mammary microenvironment acquiring an immunomodulatory
‘wound healing’ phenotype [
], which we have also
demonstrated is dependent on Stat3 [
]. Factors implicated in
acquisition of a ‘wound healing’ phenotype include
deposition of fibrillar collagen, high levels of COX-2 expression,
itself promoting lymphangiogenesis, and mammary epithelial
cell efferocytosis [
]. There is an influx of immune
cells during the second phase of involution, including mast
cells, lymphocytes, and predominantly alternatively activated
52, 53, 116, 122–124
], and postlactational
human breast tissue exhibits a transient infiltrate of high IL-10
(+) macrophages and Foxp3 (+) regulatory T cells . It is
easy to speculate that such an immunomodulatory and ‘wound
healing’ microenvironment may favour proliferation of
bacteria during involution-associated mastitis, particularly when
coupled with the presence of milk deposits, which may
provide a nidus for bacterial infections.
Intriguingly, Stat3 also regulates expression of members of
the chloride channel regulators, calcium activated, (CLCA)
family, of proteins during involution. A positive association
is observed between murine CLCA1 and CLCA2 and Stat3
activity, whilst Stat3 negatively regulates murine CLCA5, the
murine orthologue of human CLCA2 . The exact
functions of CLCA family members within the mammary gland
are yet to be determined, but their regulation by Stat3 during
the involution period may be pertinent to the pathogenesis of
involution-associated mastitis given their postulated
modulation of the innate immune response and/or potential activity as
signalling molecules [126, 127].
It is important to note that cattle are usually in the final
trimester of pregnancy during the dry period, and some
dairy goats may be pregnant, depending on the production
system . Thus, the involution process may be
markedly modulated by what we will term a ‘parallel pregnancy
signature’. Although some bovine mammary epithelial
cells undergo cell death during bovine mammary involution
], tissue regression is not notable [
]. This particular
aspect of bovine involution is therefore an important
species difference which needs to be carefully considered
when adopting a One Health viewpoint before making
In spite of the differences in progression of involution in
cattle, high levels of serum amyloid A3 expression are also
observed in bovine mammary epithelial cells during mid to
late involution and in inflammatory states [
] indicating that
the inflammatory profile of the bovine involution mammary
gland may be similar to rodent models. Experiments in which
serum amyloid A3 was infused into the mammary gland via
the teat canal suggest that serum amyloid A3 may enhance
MMP9 activity and may also reduce Staphylococcus aureus
Abrupt cessation of lactation in sheep heralds a transient
i n c r e a s e i n g l a n d c i s t e r n v o l u m e a s m e a s u r e d
ultrasonographically. Interestingly, approximately one week
after weaning, milk within the gland cistern exhibits
ultrasonographic evidence of clotting and is interpreted to be
gradually resorbed, resulting in a reduction in gland cistern volume
]. Again, it is possible that accumulation of milk within
the gland cistern may represent a potential nidus for infection,
particularly if there is compromise to the innate immune
defences of the teat canal such as through mechanical injury.
Similar to murine models, the ovine mammary gland also
exhibits involution-associated mammary epithelial cell death,
efferocytosis mediated by macrophages and mammary
epithelial cells, and ultrasonographic evidence of matrix
Importance of the Dry Period in Dairy Ruminants
There is a bi-directional relationship between the dry period
and mammary health and the dry period represents a time
utilised to cure cows or small ruminants from subclinical
mammary infections prior to the next lactation cycle,
concomitantly improving milk quality [
]. The nonlactating
period between drying off and parturition is also an important
period of renewal in dairy animals, with the first thirty days of
the dry period considered to represent a phase of active
involution, and the subsequent thirty days a period of cellular
Mouse Models of Mastitis
Mouse models of mastitis provide a tractable system in
which the mammary microenvironment can be manipulated
in a controlled manner and therefore offer a model in which
the effects of new interventions can be more readily
]. However, some protocols involve weaning
of offspring at the time of mastitis induction which causes
induction of involution . This may not recapitulate the
changes in the breast microenvironment, for example
lactating women are often advised to continued breastfeeding
where feasible and clinically indicated [
]. In addition,
anatomical differences should also be considered. For
instance, as discussed above, the mammary stroma varies
between humans, ruminants, rabbits and laboratory
rodents, and so extrapolation between species requires
prudence and an appreciation of such differences. There may
be elements of disease pathogenesis in which spontaneous
mastitis in large animals more closely recapitulates the
condition in humans, and as such study of ruminant mastitis
may yield particularly useful insights.
Clearly the environmental conditions in which humans, dairy
ruminants, and rabbits live frequently vary considerably [
and it has been suggested that this factor may militate against
the utility of a One Health approach. The conclusion of this
review is certainly not the view that a One Health focus holds
the answer to every outstanding question regarding the
pathogenesis and treatment of mastitis in humans and animals, and
as we have stated throughout, an awareness of species
differences, as well as similarities, is of paramount importance.
However, as one of many ‘tools in the armoury’ of those
involved in mastitis research, maintaining a One Health
perspective opens up additional opportunities and possibilities.
Mastitis is a painful condition, and at the heart of any
scientific discussion about this condition should be an approach
mindful of the potential psychological effect of mastitis on the
human mother and infant, and the likely welfare impact on
production and laboratory animals. A One Health mindset will
foster cross-fertilization of ideas between biomedical
scientists, whether trained in basic, medical or veterinary sciences,
and may offer the chance to expedite progress in this
important field of research.
Materials and Methods for Unpublished
Rabbits were shot for population management and
cadavers were donated to the anatomic pathology service
of the Department of Veterinary Medicine, University of
Cambridge, for research and teaching purposes. At the
time of post mortem examination, rabbits were weighed
to assess maturity [
]. Macroscopic post mortem
assessment included examination of the mammary glands
and reproductive tract. Where appropriate, stage of
pregnancy was determined by assessment of foetal
crownrump measurement. Mammary tissue from cattle and
sheep was collected from ruminants examined by the
anatomic pathology service of the Department of
Veterinary Medicine, University of Cambridge, with
owner consent for collection of tissues for research
Histology and Immunohistochemisty
Mammary tissue was collected in 10% neutral-buffered
formalin, and was subsequently processed and sectioned.
Staining with haematoxylin and eosin followed standard
histological methodology. Immunohistochemical (IHC)
staining for CD3 (dilution 1:150; mouse monoclonal,
clone F7.2.38, Dako Pathology/Agilent Technologies
LDA UK, Cheadle, Cheshire, UK) and CD20 (dilution
1:400; rabbit polyclonal, ThermoFisher Scientific, 168
Third Avenue, Waltham, MA. USA 02451) followed a
routine protocol using an automated IHC system (Dako
Acknowledgements KH’s research is funded by the British Veterinary
Association Animal Welfare Foundation Norman Hayward Fund
(NHF_2016_03_KH). Research in CJW’s laboratory is funded by the
Medical Research Council (MR/J001023/1). The authors would like to
thank Ms. V. Owenson of the Department of Veterinary Medicine,
University of Cambridge, for her excellent technical expertise in the
preparation of tissue sections. The authors gratefully acknowledge the Ethics
and Welfare Committee of the Department of Veterinary Medicine,
University of Cambridge for their review of the study plans relating to
the post mortem collection of ruminant mammary tissue for research use
(reference: CR223) and for the use of wild rabbit cadavers for the study of
mammary gland biology (reference: CR240). We apologise to all
investigators whose work could not be cited owing to space limitations.
Open Access This article is distributed under the terms of the Creative
C o m m o n s A t t r i b u t i o n 4 . 0 I n t e r n a t i o n a l L i c e n s e ( h t t p : / /
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
1. Amir LH . Managing common breastfeeding problems in the community . BMJ . 2014 ; 348 :g2954. https://doi.org/10.1136/bmj. g2954.
2. Schwartz K , D'Arcy HJ , Gillespie B , Bobo J , Longeway M , Foxman B . Factors associated with weaning in the first 3 months postpartum . J Fam Pract . 2002 ; 51 ( 5 ): 439 - 44 .
3. Sun K , Chen M , Yin Y , Wu L , Gao L . Why Chinese mothers stop breastfeeding: Mothers' self-reported reasons for stopping during the first six months . J Child Health Care . 2017 ; 21 ( 3 ): 353 - 63 . https://doi.org/10.1177/1367493517719160.
4. Victora CG , Bahl R , Barros AJ , Franca GV , Horton S , Krasevec J , et al. Breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect . Lancet . 2016 ; 387 ( 10017 ): 475 - 90 . https://doi.org/10.1016/S0140- 6736 ( 15 ) 01024 - 7 .
5. Tuaillon E , Viljoen J , Dujols P , Cambonie G , Rubbo PA , Nagot N , et al. Subclinical mastitis occurs frequently in association with dramatic changes in inflammatory/anti-inflammatory breast milk components . Pediatr Res . 2017 ; 81 ( 4 ): 556 - 64 . https://doi.org/10. 1038/pr. 2016 . 220 .
6. Dolan S , Field LC , Nolan AM . The role of nitric oxide and prostaglandin signaling pathways in spinal nociceptive processing in chronic inflammation . Pain . 2000 ; 86 ( 3 ): 311 - 20 .
7. Peters MD , Silveira ID , Fischer V . Impact of subclinical and clinical mastitis on sensitivity to pain of dairy cows . Animal . 2015 ; 9 ( 12 ): 2024 - 8 . https://doi.org/10.1017/S1751731115001391.
8. Kossaibati MA , Esslemont RJ . The costs of production diseases in dairy herds in England . Vet J. 1997 ; 154 ( 1 ): 41 - 51 .
9. Corpa JM , Hermanns K , Haesbrouck F. Main pathologies associated with Staphylococcus aureus infections in rabbits: a review . World Rabbit Sci . 2009 ; 17 : 115 - 25 .
10. Guerrero I , Ferrian S , Penades M , Garcia-Quiros A , Pascual JJ , Selva L , et al. Host responses associated with chronic staphylococcal mastitis in rabbits . Vet J . 2015 ; 204 ( 3 ): 338 - 44 . https://doi. org/10.1016/j.tvjl. 2015 . 03 .020.
11. Viana D , Selva L , Callanan JJ , Guerrero I , Ferrian S , Corpa JM . Strains of Staphylococcus aureus and pathology associated with chronic suppurative mastitis in rabbits . Vet J . 2011 ; 190 ( 3 ): 403 - 7 . https://doi.org/10.1016/j.tvjl. 2010 . 11 .022.
12. Rosell JM , de la Fuente LF. Culling and mortality in breeding rabbits . Prev Vet Med . 2009 ; 88 ( 2 ): 120 - 7 . https://doi.org/10. 1016/j.prevetmed. 2008 . 08 .003.
13. Segura P , Martinez J , Peris B , Selva L , Viana D , Penades JR , et al. Staphylococcal infections in rabbit does on two industrial farms . Vet Rec . 2007 ; 160 ( 25 ): 869 - 72 .
14. Sanchez JP , de la Fuente LF , Rosell JM . Health and body condition of lactating females on rabbit farms . J Anim Sci . 2012 ; 90 ( 7 ): 2353 - 61 . https://doi.org/10.2527/jas.2011- 4065 .
15. Mancinelli E , Lord B . Urogenital system and reproductive disease . In: Meredith A , Lord B , editors. BSAVA Manual of Rabbit Medicine. Gloucester: British Small Animal Veterinary Association; 2014 . p. 202 .
16. Zinsstag J , Schelling E , Waltner-Toews D , Tanner M. From "one medicine" to "one health" and systemic approaches to health and well-being . Prev Vet Med . 2011 ; 101 ( 3-4 ): 148 - 56 . https://doi.org/ 10.1016/j.prevetmed. 2010 . 07 .003.
17. Gibbs EP . The evolution of One Health: a decade of progress and challenges for the future . Vet Rec . 2014 ; 174 ( 4 ): 85 - 91 . https://doi. org/10.1136/vr.g143.
18. Lebov J , Grieger K , Womack D , Zaccaro D , Whitehead N , Kowalcyk B , et al. A framework for One Health research . One Health . 2017 ; 3 : 44 - 50 . https://doi.org/10.1016/j.onehlt. 2017 . 03 . 004.
19. Ingman WV , Glynn DJ , Hutchinson MR . Inflammatory mediators in mastitis and lactation insufficiency . J Mammary Gland Biol Neoplasia . 2014 ; 19 ( 2 ): 161 - 7 . https://doi.org/10.1007/s10911- 014-9325-9.
20. Cooper A. On the anatomy of the breast . 1840 .
21. Schlafer DH , Foster RA . Female Genital System . Jubb, Kennedy, and Palmer's Pathology of Domestic Animals . St. Louis: Elsevier; 2016 .
22. Ramsay DT , Kent JC , Hartmann RA , Hartmann PE . Anatomy of the lactating human breast redefined with ultrasound imaging . J Anat . 2005 ; 206 ( 6 ): 525 - 34 . https://doi.org/10.1111/j.1469- 7580 . 2005 . 00417 .x.
23. Love SM , Barsky SH . Anatomy of the nipple and breast ducts revisited . Cancer . 2004 ; 101 ( 9 ): 1947 - 57 . https://doi.org/10.1002/ cncr.20559.
24. Rowson AR , Daniels KM , Ellis SE , Hovey RC . Growth and development of the mammary glands of livestock: a veritable barnyard of opportunities . Semin Cell Dev Biol . 2012 ; 23 ( 5 ): 557 - 66 . https://doi.org/10.1016/j.semcdb. 2012 . 03 .018.
25. Cowie AT . Proceedings: Overview of the mammary gland . J Invest Dermatol . 1974 ; 63 ( 1 ): 2 - 9 .
26. Calvert DT , Knight CH , Peaker M. Milk accumulation and secretion in the rabbit . Q J Exp Physiol . 1985 ; 70 ( 3 ): 357 - 63 .
27. Lossi L , D'Angelo L , De Girolamo P , Merighi A . Anatomical features for an adequate choice of experimental animal model in biomedicine: II. Small laboratory rodents, rabbit, and pig . Ann Anat. 2016 ; 204 : 11 - 28 . https://doi.org/10.1016/j.aanat. 2015 . 10 .002.
28. Cowin P , Wysolmerski J . Molecular mechanisms guiding embryonic mammary gland development . Cold Spring Harb Perspect Biol . 2010 ; 2 ( 6 ):a003251. https://doi.org/10.1101/cshperspect. a003251.
29. Macias H , Hinck L . Mammary gland development . Wiley Interdiscip Rev Dev Biol . 2012 ; 1 ( 4 ): 533 - 57 . https://doi.org/10. 1002/wdev.35.
30. Propper AY , Howard BA , Veltmaat JM . Prenatal morphogenesis of mammary glands in mouse and rabbit . J Mammary Gland Biol Neoplasia . 2013 ; 18 ( 2 ): 93 - 104 . https://doi.org/10.1007/s10911- 013-9298-0.
31. Gusterson BA , Stein T . Human breast development . Semin Cell Dev Biol . 2012 ; 23 ( 5 ): 567 - 73 . https://doi.org/10.1016/j.semcdb. 2012 . 03 .013.
32. Musumeci G , Castrogiovanni P , Szychlinska MA , Aiello FC , Vecchio GM , Salvatorelli L , et al. Mammary gland: From embryogenesis to adult life . Acta Histochem . 2015 ; 117 ( 4-5 ): 379 - 85 . https://doi.org/10.1016/j.acthis. 2015 . 02 .013.
33. Biggs A. Update on dry cow therapy 1. antibiotic v non-antibiotic approaches . In Practice . 2017 ; 39 : 328 - 33 .
34. Brooker BE . An ultrastructural study of the sinus epithelium in the mammary gland of the lactating ewe . J Anat . 1984 ; 138 (Pt 2): 287 - 96 .
35. Going JJ , Moffat DF . Escaping from Flatland: clinical and biological aspects of human mammary duct anatomy in three dimensions . J Pathol . 2004 ; 203 ( 1 ): 538 - 44 . https://doi.org/10.1002/ path.1556.
36. Nissan N , Furman-Haran E , Shapiro-Feinberg M , Grobgeld D , D egan i H. Mo ni tori ng I n-Vi vo the Ma mma ry Gla nd Microstructure during Morphogenesis from Lactation to PostWeaning Using Diffusion Tensor MRI . J Mammary Gland Biol Neoplasia . 2017 ; https://doi.org/10.1007/s10911-017-9383-x.
37. Hovey RC , McFadden TB , Akers RM . Regulation of mammary gland growth and morphogenesis by the mammary fat pad: a species comparison . J Mammary Gland Biol Neoplasia . 1999 ; 4 ( 1 ): 53 - 68 .
38. Beaudry KL , Parsons CL , Ellis SE , Akers RM . Localization and quantitation of macrophages, mast cells, and eosinophils in the developing bovine mammary gland . J Dairy Sci . 2016 ; 99 ( 1 ): 796 - 804 . https://doi.org/10.3168/jds.2015- 9972 .
39. Dasari P , Sharkey DJ , Noordin E , Glynn DJ , Hodson LJ , Chin PY , et al. Hormonal regulation of the cytokine microenvironment in the mammary gland . J Reprod Immunol . 2014 ; 106 : 58 - 66 . https:// doi.org/10.1016/j.jri. 2014 . 07 .002.
40. Need EF , Atashgaran V , Ingman WV , Dasari P . Hormonal regulation of the immune microenvironment in the mammary gland . J Mammary Gland Biol Neoplasia . 2014 ; 19 ( 2 ): 229 - 39 . https://doi. org/10.1007/s10911-014-9324-x.
41. Nickerson SC . Immune mechanisms of the bovine udder: an overview . J Am Vet Med Assoc . 1985 ; 187 ( 1 ): 41 - 5 .
42. Ezzat Alnakip M , Quintela-Baluja M , Bohme K , Fernandez-No I , Caamano-Antelo S , Calo-Mata P , et al. The Immunology of Mammary Gland of Dairy Ruminants between Healthy and Inflammatory Conditions . J Vet Med . 2014 ; 2014 :659801. https:// doi.org/10.1155/ 2014 /659801.
43. Oviedo-Boyso J , Valdez-Alarcon JJ , Cajero-Juarez M , OchoaZarzosa A , Lopez-Meza JE , Bravo-Patino A , et al. Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis . J Inf Secur . 2007 ; 54 ( 4 ): 399 - 409 . https://doi.org/ 10.1016/j.jinf. 2006 . 06 .010.
44. Nickerson SC , Akers RM . Mammary gland anatomy . In: Fuquay JW , editor. Encyclopedia of Dairy Sciences. 2nd ed.: Elsevier; 2011 . p. 328 - 37 .
45. Wagener MG , Leonhard-Marek S , Hager JD , Pfarrer C. CD117- and vimentin-positive telocytes in the bovine teat sphincter . Anat Histol Embryol . 2018 ; https://doi.org/10.1111/ahe.12347.
46. Vesterinen HM , Corfe IJ , Sinkkonen V , Iivanainen A , Jernvall J , Laakkonen J . Teat Morphology Characterization With 3D Imaging. Anat Rec (Hoboken) . 2015 ; 298 ( 7 ): 1359 - 66 . https:// doi.org/10.1002/ar.23091.
47. Lind M , Sipka AS , Schuberth HJ , Blutke A , Wanke R , SauterLouis C , et al. Location-specific expression of chemokines, TNFalpha and S100 proteins in a teat explant model . Innate Immun . 2015 ; 21 ( 3 ): 322 - 31 . https://doi.org/10.1177/1753425914539820.
48. Nickerson SC , Pankey JW . Cytologic observations of the bovine teat end . Am J Vet Res . 1983 ; 44 ( 8 ): 1433 - 41 .
49. Mavrogianni VS , Cripps PJ , Brooks H , Taitzoglou IA , Fthenakis GC . Presence of subepithelial lymphoid nodules in the teat of ewes . Anat Histol Embryol . 2007 ; 36 ( 3 ): 168 - 71 . https://doi.org/ 10.1111/j.1439- 0264 . 2006 . 00720 .x.
50. Mavrogianni VS , Fthenakis GC . Clinical, bacteriological, cytological and pathological features of teat disorders in ewes . J Vet Med A Physiol Pathol Clin Med . 2007 ; 54 ( 4 ): 219 - 23 . https://doi. org/10.1111/j.1439- 0442 . 2007 . 00874 .x.
51. Gelasakis AI , Mavrogianni VS , Petridis IG , Vasileiou NG , Fthenakis GC . Mastitis in sheep-The last 10 years and the future of research . Vet Microbiol . 2015 ; 181 ( 1-2 ): 136 - 46 . https://doi. org/10.1016/j.vetmic. 2015 . 07 .009.
52. Stein T , Morris JS , Davies CR , Weber-Hall SJ , Duffy MA , Heath VJ , et al. Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3 . Breast Cancer Res . 2004 ; 6 ( 2 ): R75 - 91 . https://doi.org/10.1186/bcr753.
53. Hughes K , Wickenden JA , Allen JE , Watson CJ . Conditional deletion of Stat3 in mammary epithelium impairs the acute phase response and modulates immune cell numbers during postlactational regression . J Pathol . 2012 ; 227 ( 1 ): 106 - 17 . https://doi. org/10.1002/path.3961.
54. Deng Z , Shahid M , Zhang L , Gao J , Gu X , Zhang S , et al. An Investigation of the Innate Immune Response in Bovine Mammary Epithelial Cells Challenged by Prototheca zopfii . Mycopathologia . 2016 ; 181 ( 11 -12): 823 - 32 . https://doi.org/10. 1007/s11046-016-0053-0.
55. Bulgari O , Dong X , Roca AL , Caroli AM , Loor JJ . Innate immune responses induced by lipopolysaccharide and lipoteichoic acid in primary goat mammary epithelial cells . J Anim Sci Biotechnol . 2017 ; 8 : 29 . https://doi.org/10.1186/s40104-017-0162-8.
56. Henson PM . Cell Removal: Efferocytosis. Annu Rev Cell Dev Biol . 2017 ; 33 : 127 - 44 . https://doi.org/10.1146/annurev-cellbio111315- 125315 .
57. Teplova I , Lozy F , Price S , Singh S , Barnard N , Cardiff RD , et al. ATG proteins mediate efferocytosis and suppress inflammation in mammary involution . Autophagy . 2013 ; 9 ( 4 ): 459 - 75 . https://doi. org/10.4161/auto.23164.
58. Sandahl M , Hunter DM , Strunk KE , Earp HS , Cook RS . Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation . BMC Dev Biol . 2010 ; 10 : 122 . https://doi.org/10.1186/ 1471 -213X- 10 -122.
59. Tatarczuch L , Philip C , Lee CS . Involution of the sheep mammary gland . J Anat . 1997 ; 190 (Pt 3): 405 - 16 .
60. Sargeant TJ , Lloyd-Lewis B , Resemann HK , Ramos-Montoya A , Skepper J , Watson CJ . Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization . Nat Cell Biol . 2014 ; 16 ( 11 ): 1057 - 68 . https://doi.org/10.1038/ncb3043.
61. Sordillo LM , Streicher KL . Mammary gland immunity and mastitis susceptibility . J Mammary Gland Biol Neoplasia . 2002 ; 7 ( 2 ): 135 - 46 .
62. Guimaraes JLB , Brito M , Lange CC , Silva MR , Ribeiro JB , Mendonca LC , et al. Estimate of the economic impact of mastitis: A case study in a Holstein dairy herd under tropical conditions . Prev Vet Med . 2017 ; 142 : 46 - 50 . https://doi.org/10.1016/j. prevetmed. 2017 . 04 .011.
63. Flores M , Filteau S . Effect of lactation counselling on subclinical mastitis among Bangladeshi women . Ann Trop Paediatr. 2002 ; 22 ( 1 ): 85 - 8 . https://doi.org/10.1179/027249302125000210.
64. Garofalo R. Cytokines in human milk . J Pediatr . 2010 ; 156 ( 2 Suppl) : S36 - 40 . https://doi.org/10.1016/j.jpeds. 2009 . 11 .019.
65. Kvist LJ . Diagnostic methods for mastitis in cows are not appropriate for use in humans: commentary . Int Breastfeed J . 2016 ; 11 :2. https://doi.org/10.1186/s13006-016-0061-1.
66. Willumsen JF , Filteau SM , Coutsoudis A , Uebel KE , Newell ML , Tomkins AM . Subclinical mastitis as a risk factor for motherinfant HIV transmission . Adv Exp Med Biol . 2000 ; 478 : 211 - 23 . https://doi.org/10.1007/0-306-46830-1_ 19 .
67. Goncalves JL , Lyman RL , Hockett M , Rodriguez R , Dos Santos MV , Anderson KL . Using milk leukocyte differentials for diagnosis of subclinical bovine mastitis . J Dairy Res . 2017 ; 84 ( 3 ): 309 - 17 . https://doi.org/10.1017/S0022029917000267.
68. Flores-Quijano ME , Cordova A , Contreras-Ramirez V , FariasHernandez L , Cruz Tolentino M , Casanueva E . Risk for postpartum depression, breastfeeding practices, and mammary gland permeability . J Hum Lact . 2008 ; 24 ( 1 ): 50 - 7 . https://doi.org/10.1177/ 0890334407310587.
69. Truchet S , Honvo-Houeto E . Physiology of milk secretion . Best Pract Res Clin Endocrinol Metab . 2017 ; 31 ( 4 ): 367 - 84 . https://doi. org/10.1016/j.beem. 2017 . 10 .008.
70. McManaman JL , Neville MC . Mammary physiology and milk secretion . Adv Drug Deliv Rev . 2003 ; 55 ( 5 ): 629 - 41 .
71. Pang WW , Hartmann PE . Initiation of human lactation: secretory differentiation and secretory activation . J Mammary Gland Biol Neoplasia . 2007 ; 12 ( 4 ): 211 - 21 . https://doi.org/10.1007/s10911- 007-9054-4.
72. Nguyen DA , Parlow AF , Neville MC . Hormonal regulation of tight junction closure in the mouse mammary epithelium during the transition from pregnancy to lactation . J Endocrinol . 2001 ; 170 ( 2 ): 347 - 56 .
73. Neville MC . Anatomy and physiology of lactation . Pediatr Clin N Am . 2001 ; 48 ( 1 ): 13 - 34 .
74. Singh M , Yadav P , Sharma A , Garg VK , Mittal D. Estimation of Mineral and Trace Element Profile in Bubaline Milk Affected with Subclinical Mastitis . Biol Trace Elem Res . 2017 ; 176 ( 2 ): 305 - 10 . https://doi.org/10.1007/s12011-016-0842-9.
75. Filteau SM , Lietz G , Mulokozi G , Bilotta S , Henry CJ , Tomkins AM . Milk cytokines and subclinical breast inflammation in Tanzanian women: effects of dietary red palm oil or sunflower oil supplementation . Immunology . 1999 ; 97 ( 4 ): 595 - 600 .
76. Filteau SM , Rice AL , Ball JJ , Chakraborty J , Stoltzfus R , de Francisco A , et al. Breast milk immune factors in Bangladeshi women supplemented postpartum with retinol or beta-carotene . Am J Clin Nutr . 1999 ; 69 ( 5 ): 953 - 8 .
77. Rasmussen LB , Hansen DH , Kaestel P , Michaelsen KF , Friis H , Larsen T. Milk enzyme activities and subclinical mastitis among women in Guinea-Bissau . Breastfeed Med . 2008 ; 3 ( 4 ): 215 - 9 . https://doi.org/10.1089/bfm. 2007 . 0035 .
78. Karthikeyan A , Radhika G , Aravindhakshan TV , Anilkumar K. Expression Profiling of Innate Immune Genes in Milk Somatic Cells During Subclinical Mastitis in Crossbred Dairy Cows . Anim Biotechnol . 2016 ; 27 ( 4 ): 303 - 9 . https://doi.org/10.1080/ 10495398. 2016 . 1184676 .
79. Albenzio M , Santillo A , Caroprese M , Ruggieri D , Ciliberti M , Sevi A . Immune competence of the mammary gland as affected by somatic cell and pathogenic bacteria in ewes with subclinical mastitis . J Dairy Sci . 2012 ; 95 ( 7 ): 3877 - 87 . https://doi.org/10.3168/jds. 2012- 5357 .
80. Nussenblatt V , Lema V , Kumwenda N , Broadhead R , Neville MC , Taha TE , et al. Epidemiology and microbiology of subclinical mastitis among HIV-infected women in Malawi . Int J STD A I D S . 2 0 0 5 ; 1 6 ( 3 ) : 2 2 7 - 3 2 . h t t p s : / / d o i . o rg / 1 0 . 1 2 5 8 / 0956462053420248.
81. Nussenblatt V , Kumwenda N , Lema V , Quinn T , Neville MC , Broadhead R , et al. Effect of antibiotic treatment of subclinical mastitis on human immunodeficiency virus type 1 RNA in human milk . J Trop Pediatr . 2006 ; 52 ( 5 ): 311 - 5 . https://doi.org/10.1093/ tropej/fml011.
82. Highland MA . Small Ruminant Lentiviruses: Strain Variation, Viral Tropism, and Host Genetics Influence Pathogenesis . Vet P a t h o l . 2 0 1 7 ; 5 4 ( 3 ) : 3 5 3 - 4 . h t t p s : / / d o i . o r g / 1 0 . 11 7 7 / 0300985817695517.
83. Martin R , Heilig HG , Zoetendal EG , Jimenez E , Fernandez L , Smidt H , et al. Cultivation-independent assessment of the bacterial diversity of breast milk among healthy women . Res Microbiol . 2007 ; 158 ( 1 ): 31 - 7 . https://doi.org/10.1016/j.resmic. 2006 . 11 .004.
84. Martin R , Heilig GH , Zoetendal EG , Smidt H , Rodriguez JM . Diversity of the Lactobacillus group in breast milk and vagina of healthy women and potential role in the colonization of the infant gut . J Appl Microbiol . 2007 ; 103 ( 6 ): 2638 - 44 . https://doi.org/10. 1111/j.1365- 2672 . 2007 . 03497 .x.
85. Jimenez E , de Andres J , Manrique M , Pareja-Tobes P , Tobes R , Martinez-Blanch JF , et al. Metagenomic Analysis of Milk of Healthy and Mastitis-Suffering Women . J Hum Lact . 2015 ; 31 ( 3 ): 406 - 15 . https://doi.org/10.1177/0890334415585078.
86. Angelopoulou A , Field D , Ryan CA , Stanton C , Hill C , Ross RP . The microbiology and treatment of human mastitis . Med Microbiol Immunol . 2018 ; 207 ( 2 ): 83 - 94 . https://doi.org/10.1007/ s00430-017-0532-z.
87. Osterman KL , Rahm VA . Lactation mastitis: bacterial cultivation of breast milk, symptoms, treatment, and outcome . J Hum Lact. 2 0 0 0 ; 1 6 ( 4 ) : 2 9 7 - 3 0 2 . h t t p s : / / d o i . o r g / 1 0 . 1 1 7 7 / 089033440001600405.
88. Kvist LJ , Larsson BW , Hall-Lord ML , Steen A , Schalen C . The role of bacteria in lactational mastitis and some considerations of the use of antibiotic treatment . Int Breastfeed J . 2008 ; 3:6 . https:// doi.org/10.1186/ 1746 -4358-3-6.
89. Turner PV , Brash ML , Smith DA . Rabbits. In: Turner PV , Brash ML , Smith DA , editors. Pathology of Small Mammal Pets . Hoboken: John Wiley & Sons, Inc; 2018 . p. 11 .
90. Wesche P. Clinical Pathology . In: Meredith A , Lord B , editors. BSAVA Manual of Rabbit Medicine. Gloucester: British Small Animal Veterinary Association; 2014 . p. 125 - 6 .
91. Sordillo LM , Doymaz MZ , Oliver SP . Distribution of immunoglobulin-bearing leukocytes in bovine mammary tissue infected chronically with Staphylococcus aureus . Zentralbl Veterinarmed B . 1990 ; 37 ( 6 ): 473 - 6 .
92. Petzl W , Gunther J , Muhlbauer K , Seyfert HM , Schuberth HJ , Hussen J , et al. Early transcriptional events in the udder and teat after intra-mammary Escherichia coli and Staphylococcus aureus challenge . Innate Immun . 2016 ; 22 ( 4 ): 294 - 304 . https://doi.org/10. 1177/1753425916640057.
93. Whelehan CJ , Meade KG , Eckersall PD , Young FJ , O'Farrelly C. Experimental Staphylococcus aureus infection of the mammary gland induces region-specific changes in innate immune gene expression . Vet Immunol Immunopathol . 2011 ; 140 ( 3-4 ): 181 - 9 . https://doi.org/10.1016/j.vetimm. 2010 . 11 .013.
94. Wellnitz O , Arnold ET , Bruckmaier RM . Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland . J Dairy Sci . 2011 ; 94 ( 11 ): 5405 - 12 . https://doi. org/10.3168/jds.2010- 3931 .
95. Lutzow YC , Donaldson L , Gray CP , Vuocolo T , Pearson RD , Reverter A , et al. Identification of immune genes and proteins involved in the response of bovine mammary tissue to Staphylococcus aureus infection . BMC Vet Res . 2008 ; 4 : 18 . https://doi.org/10.1186/ 1746 -6148-4-18.
96. Gunther J , Petzl W , Bauer I , Ponsuksili S , Zerbe H , Schuberth HJ , et al. Differentiating Staphylococcus aureus from Escherichia coli mastitis: S. aureus triggers unbalanced immune-dampening and host cell invasion immediately after udder infection . Sci Rep . 2017 ; 7 ( 1 ): 4811 . https://doi.org/10.1038/s41598-017-05107-4.
97. Breyne K , Steenbrugge J , Demeyere K , Vanden Berghe T , Meyer E. Preconditioning with Lipopolysaccharide or Lipoteichoic Acid Protects against Staphylococcus aureus Mammary Infection in Mice . Front Immunol . 2017 ; 8 : 833 . https://doi.org/10.3389/ fimmu. 2017 . 00833 .
98. Zhao S , Gao Y , Xia X , Che Y , Wang Y , Liu H , et al. TGF-beta1 promotes Staphylococcus aureus adhesion to and invasion into bovine mammary fibroblasts via the ERK pathway . Microb Pathog . 2017 ; 106 : 25 - 9 . https://doi.org/10.1016/j.micpath. 2017 . 01 .044.
99. Wagner SA , Jones DE , Apley MD . Effect of endotoxic mastitis on epithelial cell numbers in the milk of dairy cows . Am J Vet Res . 2009 ; 70 ( 6 ): 796 - 9 . https://doi.org/10.2460/ajvr.70.6.796.
100. Buitenhuis B , Rontved CM , Edwards SM , Ingvartsen KL , Sorensen P. In depth analysis of genes and pathways of the mammary gland involved in the pathogenesis of bovine Escherichia coli-mastitis . BMC Genomics . 2011 ; 12 : 130 . https://doi.org/10. 1186/ 1471 -2164-12-130.
101. Ferreira AM , Bislev SL , Bendixen E , Almeida AM . The mammary gland in domestic ruminants: a systems biology perspective . J Proteome . 2013 ; 94 : 110 - 23 . https://doi.org/10.1016/j.jprot. 2013 . 09 .012.
102. Glynn DJ , Hutchinson MR , Ingman WV . Toll-like receptor 4 regulates lipopolysaccharide-induced inflammation and lactation insufficiency in a mouse model of mastitis . Biol Reprod . 2014 ; 90 ( 5 ): 91 . https://doi.org/10.1095/biolreprod.114.117663.
103. Green MJ , Bradley AJ , Medley GF , Browne WJ . Cow, farm, and herd management factors in the dry period associated with raised somatic cell counts in early lactation . J Dairy Sci . 2008 ; 91 ( 4 ): 1403 - 15 . https://doi.org/10.3168/jds.2007- 0621 .
104. Mavrogianni VS , Menzies PI , Fragkou IA , Fthenakis GC . Principles of mastitis treatment in sheep and goats . Vet Clin North Am Food Anim Pract . 2011 ; 27 ( 1 ): 115 - 20 . https://doi.org/ 10.1016/j.cvfa. 2010 . 10 .010.
105. Bergonier D , de Cremoux R , Rupp R , Lagriffoul G , Berthelot X . Mastitis of dairy small ruminants . Vet Res . 2003 ; 34 ( 5 ): 689 - 716 . https://doi.org/10.1051/vetres:2003030.
106. Zaragoza R , Garcia-Trevijano ER , Lluch A , Ribas G , Vina JR . Involvement of Different networks in mammary gland involution after the pregnancy/lactation cycle: Implications in breast cancer . IUBMB Life . 2015 ; 67 ( 4 ): 227 - 38 . https://doi.org/10.1002/iub. 1365.
107. Hughes K , Watson CJ . The spectrum of STAT functions in mammary gland development . JAKSTAT . 2012 ; 1 ( 3 ): 151 - 8 . https:// doi.org/10.4161/jkst.19691.
108. Hughes K , Watson CJ . The role of Stat3 in mammary gland involution: cell death regulator and modulator of inflammation . Horm Mol Biol Clin Investig . 2012 ; 10 ( 1 ): 211 - 5 . https://doi.org/10. 1515/hmbci-2012-0008.
109. Chapman RS , Lourenco PC , Tonner E , Flint DJ , Selbert S , Takeda K , et al. Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3 . Genes Dev . 1999 ; 13 ( 19 ): 2604 - 16 .
110. Humphreys RC , Bierie B , Zhao L , Raz R , Levy D , Hennighausen L. Deletion of Stat3 blocks mammary gland involution and extends functional competence of the secretory epithelium in the absence of lactogenic stimuli . Endocrinology . 2002 ; 143 ( 9 ): 3641 - 50 . https://doi.org/10.1210/en.2002- 220224 .
111. Kreuzaler PA , Staniszewska AD , Li W , Omidvar N , Kedjouar B , Turkson J , et al. Stat3 controls lysosomal-mediated cell death in vivo . Nat Cell Biol . 2011 ; 13 ( 3 ): 303 - 9 . https://doi.org/10. 1038/ncb2171.
112. Li M , Liu X , Robinson G , Bar-Peled U , Wagner KU , Young WS , et al. Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution . Proc Natl Acad Sci U S A . 1997 ; 94 ( 7 ): 3425 - 30 .
113. Schere-Levy C , Buggiano V , Quaglino A , Gattelli A , Cirio MC , Piazzon I , et al. Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution . Exp Cell Res . 2003 ; 282 ( 1 ): 35 - 47 .
114. Kritikou EA , Sharkey A , Abell K , Came PJ , Anderson E , Clarkson RW , et al. A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland . Development . 2003 ; 130 ( 15 ): 3459 - 68 .
115. Lund LR , Romer J , Thomasset N , Solberg H , Pyke C , Bissell MJ , et al. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways . Development . 1996 ; 122 ( 1 ): 181 - 93 .
116. O 'Brien J , Lyons T , Monks J , Lucia MS , Wilson RS , Hines L , et al. Alternatively activated macrophages and collagen remodeling characterize the postpartum involuting mammary gland across species . Am J Pathol . 2010 ; 176 ( 3 ): 1241 - 55 . https://doi.org/10. 2353/ajpath. 2010 . 090735 .
117. O 'Brien J , Martinson H , Durand-Rougely C , Schedin P. Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution . Development . 2012 ; 139 ( 2 ): 269 - 75 . https://doi.org/10.1242/dev.071696.
118. Stanford JC , Young C , Hicks D , Owens P , Williams A , Vaught DB , et al. Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution . J Clin Invest . 2014 ; 124 ( 11 ): 4737 - 52 . https://doi.org/10.1172/JCI76375.
119. Lyons TR , O'Brien J , Borges VF , Conklin MW , Keely PJ , Eliceiri KW , et al. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2 . Nat Med . 2011 ; 17 ( 9 ): 1109 - 15 . https://doi.org/10.1038/nm.2416.
120. Lyons TR , Borges VF , Betts CB , Guo Q , Kapoor P , Martinson HA , et al. Cyclooxygenase-2 -dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer . J Clin Invest . 2014 ; 124 ( 9 ): 3901 - 12 . https://doi.org/10.1172/JCI73777.
Hugo HJ , Saunders C , Ramsay RG , Thompson EW . New Insights on COX-2 in Chronic Inflammation Driving Breast Cancer Growth and Metastasis. J Mammary Gland Biol Neoplasia . 2015 ; 20 ( 3-4 ): 109 - 19 . https://doi.org/10.1007/s10911-015-9333-4.
Clarkson RW , Wayland MT , Lee J , Freeman T , Watson CJ . Gene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in postlactational regression . Breast Cancer Res . 2004 ; 6 ( 2 ): R92 - 109 .
Alterations in mast cell frequency and relationship to angiogenesis in the rat mammary gland during windows of physiologic tissue remodeling . Dev Dyn . 2012 ; 241 ( 5 ): 890 - 900 . https://doi.org/10.
2015 ; 136 ( 8 ): 1803 - 13 . https://doi.org/10.1002/ijc.29181.
Hughes K , Blanck M , Pensa S , Watson CJ . Stat3 modulates chloride channel accessory protein expression in normal and neoplastic mammary tissue . Cell Death Dis . 2016 ; 7 ( 10 ):e2398. https://doi.
org/10 .1038/cddis. 2016 . 302 .
mCLCA3 modulates IL-17 and CXCL-1 induction and leukocyte recruitment in murine Staphylococcus aureus pneumonia . PLoS One . 2014 ; 9 ( 7 ):e102606. https://doi.org/10.1371/journal.pone.
Dietert K , Mundhenk L , Erickson NA , Reppe K , Hocke AC , Kummer W , et al. Murine CLCA5 is uniquely expressed in distinct niches of airway epithelial cells . Histochem Cell Biol .
2015 ; 143 ( 3 ): 277 - 87 . https://doi.org/10.1007/s00418-014-1279-x.
Capuco AV , Akers RM . Mammary involution in dairy animals . J Mammary Gland Biol Neoplasia . 1999 ; 4 ( 2 ): 137 - 44 .
129. Wilde CJ , Addey CV , Li P , Fernig DG . Programmed cell death in bovine mammary tissue during lactation and involution . Exp Physiol . 1997 ; 82 ( 5 ): 943 - 53 .
130. Capuco AV , Akers RM , Smith JJ . Mammary growth in Holstein cows during the dry period: quantification of nucleic acids and histology . J Dairy Sci . 1997 ; 80 ( 3 ): 477 - 87 . https://doi.org/10. 3168/jds.S0022- 0302 ( 97 ) 75960 - 5 .
131. Molenaar AJ , Harris DP , Rajan GH , Pearson ML , Callaghan MR , Sommer L , et al. The acute-phase protein serum amyloid A3 is expressed in the bovine mammary gland and plays a role in host defence . Biomarkers . 2009 ; 14 ( 1 ): 26 - 37 . https://doi.org/10.1080/ 13547500902730714.
132. Domenech A , Pares S , Bach A , Aris A . Mammary serum amyloid A3 activates involution of the mammary gland in dairy cows . J Dairy Sci . 2014 ; 97 ( 12 ): 7595 - 605 . https://doi.org/10.3168/jds. 2014- 8403 .
133. Petridis IG , Gouletsou PG , Barbagianni MS , Amiridis GS , Brozos C , Valasi I , et al. Ultrasonographic findings in the ovine udder during involution . J Dairy Res . 2014 ; 81 ( 3 ): 288 - 96 . https://doi. org/10.1017/S0022029914000223.
134. Bradley AJ , Breen JE , Payne B , Green MJ . A comparison of broad-spectrum and narrow-spectrum dry cow therapy used alone and in combination with a teat sealant . J Dairy Sci . 2011 ; 94 ( 2 ): 692 - 704 . https://doi.org/10.3168/jds.2010- 3192 .
135. Ingman WV , Glynn DJ , Hutchinson MR . Mouse models of mastitis - how physiological are they? Int Breastfeed J. 2015 ; 10 : 12 . https://doi.org/10.1186/s13006-015-0038-5.
136. Amir LH , Trupin S , Kvist LJ . Diagnosis and treatment of mastitis in breastfeeding women . J Hum Lact . 2014 ; 30 ( 1 ): 10 - 3 . https:// doi.org/10.1177/0890334413516065.
137. Contreras GA , Rodriguez JM . Mastitis: comparative etiology and epidemiology . J Mammary Gland Biol Neoplasia . 2011 ; 16 ( 4 ): 339 - 56 . https://doi.org/10.1007/s10911-011-9234-0.
138. Lello J , Boag B , Hudson PJ . The effect of single and concomitant pathogen infections on condition and fecundity of the wild rabbit (Oryctolagus cuniculus) . Int J Parasitol . 2005 ; 35 ( 14 ): 1509 - 15 . https://doi.org/10.1016/j.ijpara. 2005 . 06 .002.