BACKGROUND
mastitis, an inflammatory condition of the mammary gland, typically arises as a result of intramammary infections.
in lactating dairy cattle, mastitis results in considerable monetary losses to the dairy industry due to depressed milk yield and quality, reduced reproductive performance, treatment, and pre-mature culling  <cit> .
staphylococcus aureus is a gram-positive, opportunistic, and contagious bacterium that is one of the most prevalent causes of mastitis in both humans and cattle  <cit> .
although infection can result in obvious clinical mastitis, s. aureus often evades the immune response resulting in a sub-clinical chronic infection that can persist for the life of the animal  <cit> .
classically, s. aureus is characterized as an extracellular pathogen  <cit> .
however, an increasing number of studies elude to its ability to invade and survive within various host cell types, including mammary epithelial cells, neutrophils, and macrophages  <cit> ; this may contribute to its persistence in various s. aureus-induced diseases  <cit> .
small colony variants  of s. aureus may be particularly endowed for persistent survival by residing in host cells as they are characterized by slow-growth and atypical colony morphology, and are often over-looked by standard laboratory assays  <cit> .
it has been previously demonstrated that scvs can survive within professional and non-professional phagocytic cells  <cit> , are more persistent than typical s. aureus strains  <cit> , and are difficult to treat with conventional antibiotics  <cit> .
given that different s. aureus strains appear to utilize unique infection and survival strategies, we hypothesized that the host immune response will also vary in accordance with the infecting strain.
to elucidate potential differences in host responses to s. aureus infection, the present study was carried out to investigate the kinetic response of key cytokines in serum and casein-depleted milk, as well as serum haptoglobin over a 21-day period following intramammary infection with three strains of s. aureus.
additionally, a comparative proteomics platform was used to resolve the proteome of casein-depleted milk during acute and chronic phases of infection, and to identify differentially expressed proteins with potential immunomodulating effects.
the s. aureus strains used for intramammary infection were: the scv heba <dig>  a naturally occurring scv isolated from a dairy cow with history of chronic mastitis with atypical phenotypic properties  <cit> , the  <dig> parent strain displaying typical morphological and biochemical properties  <cit> , and newbould  <dig> , a teat skin strain that causes clinical bovine mastitis  <cit>  that is frequently used as a model for studying s. aureus-induced mastitis.
RESULTS
serum il- <dig>  ifn-γ, tgf-β <dig>  and haptoglobin response to s. aureus intramammary infection
lactating dairy cattle were challenged with one of three s. aureus strains and the local and systemic host immune responses were characterized by monitoring cytokine kinetics and using a proteomics approach to identify proteins that may play a role in host defense .
significant il- <dig> expression in the serum was not evident for any of the challenge groups until day  <dig> post-infection  .
animals challenged with newbould  <dig> and heba  <dig> exhibited significantly higher il- <dig> expression on days  <dig> and  <dig> pi relative to levels on day  <dig> .
interestingly, animals challenged with parent  <dig> showed a significant decrease in il- <dig> expression on day  <dig> , and no significant change on day  <dig> pi, relative to day  <dig>  significant differences in il- <dig> expression between the three challenge groups were observed on days  <dig> and  <dig> pi, with both newbould  <dig> and heba  <dig> challenge groups having significantly higher il- <dig> expression than the parent  <dig> group .
milk il- <dig> levels were not statistically significantly different from baseline  levels at any time during the study .
serum concentrations of ifn-γ were significantly up-regulated in the newbould  <dig> challenge group during days 1- <dig> pi relative to serum ifn-γ levels at day  <dig>  .
on day  <dig> pi however, all challenge groups showed significant increases in ifn-γ expression compared to day  <dig> .
significant differences between challenge groups occurred on days 1- <dig> pi, whereby animals challenged with newbould  <dig> had significantly higher serum ifn-γ levels compared to animals challenged with heba  <dig> .
serum tgf-β <dig> expression was significantly down-regulated in animals challenged with parent  <dig> throughout days 1- <dig> and  <dig> pi relative to day  <dig>  .
in contrast, on day  <dig> pi, the newbould  <dig> and heba  <dig> challenge groups had significantly up-regulated expression .
on day  <dig> pi, the heba  <dig> challenge group was the only group to have significantly elevated expression of tgf-β <dig> compared to levels at day  <dig> .
significant differences in tgf-β <dig> expression between the three challenge groups were observed on day  <dig> pi, with newbould  <dig> and heba  <dig> groups having significantly higher tgf-β <dig> levels compared to the parent  <dig> group , and on day  <dig> pi, with the heba  <dig> group having significantly higher tgf-β <dig> expression compared to either newbould  <dig> or parent  <dig> groups .
milk tgf-β <dig> levels were not statistically significantly different from baseline at any time during the study period .
analysis of serum revealed significant elevations in haptoglobin concentrations within the first  <dig> hours pi for all three challenge groups  ; however, linear and orthogonal polynomial contrasts did not reveal any significant differences in haptoglobin responses between any of the challenge groups.
milk il- <dig>  ifn-γ and tgf-β <dig> response to s. aureus intramammary infection
significant changes in il- <dig> and tgf-β <dig> expression were not observed in casein-depleted milk for any of the challenge groups at any time, when compared to levels on day  <dig> .
in contrast, ifn-γ concentrations were significantly increased in animals challenged with s. aureus, but the response varied with the infection strain .
for example, ifn-γ concentrations were increased in response to newbould  <dig> on days 1- <dig> and day  <dig> pi relative to levels on day  <dig> , but not day  <dig> pi.
the parent  <dig> challenge group also showed a significant increase in ifn-γ expression on days 1- <dig> pi .
no significant change in ifn-γ expression was seen for the heba  <dig> challenge group at any sampling time.
significant differences between groups occurred on days 1- <dig> and day  <dig> pi, where the newbould  <dig> challenge group had higher ifn-γ expression compared to the heba  <dig> group .
milk proteome profiling following intramammary infection with s. aureus
milk proteome analyses by 2-dimensional gel electrophoresis  revealed  <dig> protein spots that were differentially expressed on days  <dig> or  <dig> pi, relative to expression on day  <dig>  , with up-regulated expression occurring most frequently.
in the newbould  <dig> challenge group, there were  <dig> and  <dig> protein spots that were significantly induced on days  <dig> and  <dig> pi, respectively.
in the group challenged with the parent  <dig> strain,  <dig> and  <dig> protein spots were significantly induced on days  <dig> and  <dig> pi, respectively.
lastly, for the heba  <dig> challenge group, no proteins were induced on day  <dig> pi, however,  <dig> protein spots were significantly induced on day  <dig>
nb, newbould 305; p, parent 3231; heba, scv heba 3231
trmt*day denotes the treatment and day of the observed change in protein spot
cpp <dig>  component  <dig> of the proteose peptone
data are presented as the least square mean ± standard error of the mean .
direction indicates the up-or down-regulation of the protein spot on day  <dig> or  <dig> pi, compared to baseline.
spot i.d.
number refers to protein spot number assigned by the melanie software.
spots that were excised and identified by lc-ms/ms are indicated in parentheses.
of the  <dig> spots that were differentially expressed during the acute or chronic phases of infection, only  <dig> spot was significantly differentially expressed over time and between the s. aureus challenge groups based on the fdr-adjusted p-value .
this spot, and four other spots that were differentially expressed over time and between the treatment groups based on their raw p-values , were excised and subjected to protein sequencing by liquid chromatography and tandem mass spectrometry .
identification of these protein spots and their expression patterns is summarized in table  <dig> and figure  <dig>  three out of the  <dig> spots were identified as component- <dig> of the proteose peptone .
two of the spots corresponding to cpp <dig>  were up-regulated in animals challenged with parent  <dig> on day  <dig> and  <dig> pi , as well as in the heba  <dig> challenge group on day  <dig> pi, with treatment differences occurring on day  <dig> pi between newbould  <dig> and parent  <dig> or heba  <dig> challenge groups .
in contrast, cpp <dig>  was only up-regulated in the newbould  <dig> challenge group on day  <dig> pi , with significant treatment differences occurring between the newbould  <dig> and heba  <dig> challenge groups .
aion score is = 10*log where p is the probability that the observed match is a random event ;% coverage.
btheoretical  and observed  masses and isoelectric points .
discussion
the current study aimed to monitor the host immune response to s. aureus intramammary infection using three unique strains, and to identify potential candidates for targeted therapies that may modulate the host defense to s. aureus challenge.
intramammary infection with different strains of s. aureus resulted in variable cytokine responses within the bovine mammary gland and circulation, supporting our hypothesis that different strains of s. aureus prompt the activation of host immune responses that are unique to the infecting strain.
typically, an increase in milk somatic cell count  is indicative of an elevated and sustained inflammatory response caused by intramammary infection.
during acute mastitis for example, neutrophil numbers in the mammary gland increase dramatically, constituting > 90% of milk somatic cells  <cit> , whereas somatic cells in healthy glands contain < 22% neutrophils  <cit> .
cxcl-8/il- <dig> is the main tissue-derived chemoattractant for neutrophils  <cit> ; however, our observations demonstrated that the bovine il- <dig> response did not always parallel the somatic cell response during the different s. aureus intramammary infections.
somatic cell scores were significantly elevated throughout the study period, and peaked during the acute phase  in all challenge groups , however, we did not observe significant induction of il- <dig> in the casein-depleted milk at any point during the study period, and it was only detected in the serum on days  <dig> and  <dig> pi in animals challenged with newbould  <dig> and heba  <dig>  the lack of an il- <dig> response in the milk is consistent with two previously reported independent studies carried out by bannerman et al.
and riollet et al.
<cit> , suggesting that bovine il- <dig> is not the only chemokine involved in trafficking neutrophils to the infected mammary gland.
it is possible that other chemoattractants, such as complement cleavage factor 5a, il-1β, tnf-α, cxcl <dig> and  <dig>  and granulocyte-macrophage colony stimulating factor  produced by epithelial cells, macrophages and t cells, also contribute to neutrophil trafficking to the mammary gland during intramammary infection  <cit> .
for instance, neutrophil infiltration into the bovine mammary gland may be mediated by ifn-γ through the mechanism described by mcloughlin et al.
<cit>  that involves cxc chemokines other than il- <dig>  such chemokines may include cxcl <dig> and cxcl <dig>  which are constitutively expressed in bovine milk  <cit> .
in support of this, it has been demonstrated that il- <dig> and gm-csf are not active chemokines during lactation, whereas other factors such as il-1β and tnf-α are active during any stage of lactation  <cit> .
in the present study, ifn-γ was found to be highly expressed both locally, as indicated by levels in the milk, and systemically during days 1- <dig> pi in animals challenged with newbould  <dig>  which parallels previously reported results by bannerman et al.
<cit> .
serum ifn-γ levels were significantly up-regulated in all challenge groups at day  <dig> pi compared to day  <dig>  this finding may be associated with the promotion of an enhanced cell-mediated immune response that includes neutrophil and macrophage activation.
during the chronic phase of infection, differences in serum tgf-β <dig> expression between the three challenge groups were observed with notable increases being observed for the parent  <dig> and heba  <dig> groups.
milk tgf-β <dig> did not significantly differ in response between any of the challenge groups; however, a trend towards altered tgf-β <dig> expression in animals challenged with newbould  <dig> was observed, whereby the cytokine levels reached a maximum at  <dig> hours pi .
this observation is in line with a previous report of milk tgf-β <dig> kinetics upon newbould  <dig> challenge in dairy cattle  <cit> .
the role of tgf-β in the host response to intramammary infection is uncertain because tgf-β is a pleiotropic immunoregulatory cytokine, with both pro- and anti-inflammatory functions, depending on the location and activation state of the cells that it is interacting with  <cit> .
this is exemplified by the cytokine's dual actions on macrophages.
for instance, tgf-β suppresses tissue macrophage function and inflammation by down-regulating the production of chemokines and cytokines such as ifn-γ  <cit> , up-regulating the expression of il- <dig> receptor antagonist  <cit> , and suppressing the production of cytotoxic reactive oxygen and nitrogen intermediates  <cit> .
in contrast, tgf-β has also been shown to promote inflammation by inducing chemotaxis in human peripheral blood monocytes  <cit> , enhancing their ability for transendothelial migration to sites of infection  <cit> , and up-regulating the transcription of pro-inflammatory cytokines such as il-1β and tnf-α.
furthermore, tgf-β has been shown to be an important factor for the differentiation of a subset of effector th cells known as th <dig> cells, which elicit inflammatory responses during certain pathogenic infections that are not sufficiently dealt with by th <dig> or th <dig> immunity  <cit> .
lastly, regulatory cells including tregs and m <dig> macrophages also produce tgf-β in humans.
these cells help to prevent the deleterious effects of prolonged or excessive host immune responses  <cit> , and this process includes suppressing various components of the immune response such as antigen presentation by antigen presenting cells  <cit> , and promoting tissue repair by increasing the deposition of extracellular matrix at the site of tissue injury, and promoting angiogenesis  <cit> .
since tgf-β was not observed locally in the present study, it is unlikely that it was involved in repair of the mammary gland epithelium.
there is evidence to suggest that s. aureus may be able to modulate the host immune response in part by promoting tgf-β expression; this could be a mechanism utilized by the heba  <dig> strain for example, to promote its survival within the host.
in support of this, staphylococcal superantigens  have been demonstrated to induce the production of tgf-β by initiating the differentiation and expansion of tregs that down-regulate the inflammatory response  <cit> .
indeed, tgf-β has been implicated in playing a role in immunosuppression by up-regulating the expansion and/or activation of anergic regulatory t cells in the presence of s. aureus sags, which may disrupt class ii mhc antigen presentation by apcs  <cit> , limiting cell-mediated immunity.
interestingly, this immunomodulatory mechanism is also employed by leishmania as a means to suppress cell-mediated immunity and promote its intracellular survival  <cit> .
lastly, it should be noted that tgf-β expression may not necessarily denote its level of bioactivity, since a study by kehrl et al.
<cit>  demonstrated that human b cells largely express inactive tgf-β in response s. aureus cowan strain i.
overall, haptoglobin concentrations increased with time in all challenge groups during the first  <dig> days of the study period, and declined to initial levels by days  <dig> and  <dig> pi.
however, no significant differences in serum haptoglobin levels were observed between the challenge groups.
this may have been due to the chosen sampling times, as no samples were taken between days  <dig> and  <dig> pi.
it has been previously demonstrated that haptoglobin is up-regulated during the acute phase response, and that it may be used to discriminate between acute and chronic inflammation in cattle  <cit> .
the findings in the present study indicate that overall serum haptoglobin levels were not significantly different between any of the challenge groups, suggesting that haptoglobin is a sensitive marker of inflammation due to s. aureus but it lacks specificity to discriminate the host response to the three different strains of s. aureus.
it was previously demonstrated that two common mastitis-causing pathogens, escherichia coli and s. aureus, elicit different immune responses and pathogenesis  <cit> .
here, we demonstrate that the variability in immune protein response is also strain-specific, at least in the context of s. aureus.
further studies investigating the profiles of other cytokines will add to the results of the current study.
studying il- <dig> and il- <dig> for instance would provide insight into the involvement of th <dig> cells in s. aureus infections, as il- <dig> is a th <dig> cytokine and il- <dig>  along with tgf-β supports the differentiation of this th subtype  <cit> .
furthermore, il- <dig> is one of the major signals for the induction of the acute phase response  <cit> , and may therefore compliment the present haptoglobin data.
monitoring chemoattractive factors, especially those under the regulation of tgf-β , would help delineate the pro- or anti-inflammatory role of tgf-β in intramammary infection with different strains of s. aureus.
although it would have been interesting to measure il-1-β and tnf-α as well as other cytokines such as il- <dig>  il- <dig>  il- <dig>  and il- <dig> that regulate the differentiation of these t-cell subpopulations, we had limited resources to carry out the cytokine analysis, and felt that inclusion of these cyctokies was beyond the scope of the current study as the aim was to note differences in host responses to different s. aureus strains, not to determine whether tgf-β is a pro- or anti-inflammatory cytokine.
in addition to seeing unique cytokine profiles in response to the different strains of s. aureus, the present study also found a number of milk proteins to be differentially expressed at various time-points in response to infection, including host defense proteins.
staphylococcus aureus insult to the mammary gland causes an inflammatory response that is characterized by an increase in vascular permeability, allowing exudates containing cells and proteins from the vasculature to enter the mammary gland  <cit> .
additionally, resident and recruited cells produce a number of proteins that target the pathogen and contribute to the restoration of homeostasis  <cit> .
therefore, the proteome of the milk may drastically change in response to pathogenic stimuli, and analysis of these changes may offer insight into the host response during mastitis.
indeed, the present study demonstrated changes in the milk proteome during intramammary infection with s. aureus.
in order to assess protein expression in milk during the acute and chronic phases of infection, samples were evaluated on days  <dig> and  <dig> pi, respectively.
most proteins were up-regulated following newbould  <dig> and parent  <dig> challenges, especially on day  <dig> pi during the acute phase of infection.
on day  <dig> pi, the number of up-regulated proteins was dramatically reduced in the newbould  <dig> and parent  <dig> challenge groups, as compared to day  <dig> pi.
results from this study indicate that differences in milk protein expression also occur as a result of intramammary infection with different strains of s. aureus.
on day  <dig> pi for example, challenges with newbould  <dig> and parent  <dig> induced  <dig> milk proteins each, relative to day  <dig>  in contrast, significant changes in milk protein expression were not observed in response to challenge with heba  <dig> at this time.
on day  <dig> pi however,  <dig> milk proteins were induced by challenge with the parent  <dig> strain and only  <dig> protein was induced by newbould  <dig>  whereas  <dig> proteins were significantly induced by the heba  <dig> challenge.
these results are indicative of the acute pathogenic nature of the parent  <dig> and newbould  <dig> strains and the unique phenotypic properties of scvs, which may elicit a milder host response  <cit> .
they suggest a highly active initial host response to challenges with the parent  <dig> and newbould  <dig> strains, then perhaps resolution or establishment of chronic infection by day  <dig> pi.
this corresponds with the somatic cell score data from these same cows presented by atalla et al.
<cit> , whereby the number of somatic cells in the milk during the chronic phase of infection still remained significantly up-regulated relative to day  <dig>  interestingly, we were not able to detect an initial host response to the scv strain, but the induction of protein expression was demonstrated on day  <dig> pi.
this delayed and relatively low level of host response corresponds to the ability of the heba  <dig> strain to avoid recognition and persist within host cells  <cit> .
future studies should include later time points in order to determine if heba  <dig> continues to up-regulate milk protein expression.
two of the five spots corresponded to the most abundant milk proteins - β-casein precursor and β-lactoglobulin.
this finding is likely a result of incomplete depletion of these highly abundant proteins.
nonetheless, the depletion protocol was found to be sufficient to yield a comprehensive view of the milk proteome.
of the five protein spots that were subjected to identification by lc-ms/ms, only one protein, cpp <dig> also known as lactophorin, was implicated in host defense.
component  <dig> of the proteose peptone is a phosphoglycoprotein uniquely expressed in the mammary gland of lactating dairy cattle and can be found in bovine milk whey.
it has been previously suggested to have several functions that include antimicrobial activity, inhibition of lipolysis, mitogenic activity, and immunostimulation  <cit> .
it consists of seven polypeptide components, ranging in molecular weight from  <dig> to  <dig> kda  <cit> .
in the 2de gels, it occurred at three different locations ranging in mass from 15- <dig> kda and isoelectric point  from 6- <dig> , which may correspond to its various components.
in one study, the antimicrobial activity of cpp <dig> was investigated using a synthetic peptide called lactophoricin consisting of the 113- <dig> region of c-terminal of cpp <dig>  <cit> .
lactophoricin interacts with membrane phospholipids and forms voltage-dependent channels.
thus, it has been hypothesized that it may also be involved in the pore forming of natural lipid bilayers such as bacterial membranes  <cit> .
indeed, campagna's results demonstrated that this peptide has inhibitory-growth effects on a number of gram-positive and gram-negative bacteria, with a more pronounced inhibitory effect on gram-positive bacteria, including s. aureus  <cit> .
CONCLUSIONS
the present study demonstrates the complexity of the host immune protein response to various strains of s. aureus.
intramammary infection with different strains of s. aureus, elicited unique host immune protein responses, as indicated by differential expression of the cytokines il- <dig>  tgf-β, and ifn-γ in circulation and ifn-γ in casein-depleted milk.
in general, cytokines displayed heightened responses in the serum compared to the casein-depleted milk.
interestingly, the scv strain, heba  <dig>  showed increased levels of serum il- <dig>  ifn-γ, and tgf-β occurring during the later stages of infection .
whether this pattern of induction continues in later time points has yet to be determined.
using a proteomic approach, we have also demonstrated that intramammary infection with three different strains of s. aureus elicits differential protein expression in bovine milk during the acute and chronic phases of infection.
intramammary infection with newbould  <dig> and parent  <dig> strains resulted in pronounced induction of protein expression on day  <dig> pi, while the heba  <dig> strain elicited no protein induction at this time.
on day  <dig> pi, host protein activity was less pronounced for animals challenged with newbould  <dig> and parent  <dig> strains, while some induction occurred in animals challenged with heba  <dig>  lastly, cpp <dig>  a potential antimicrobial peptide that is uniquely expressed in the bovine mammary gland during lactation, appeared on our 2de gels as three different spots, which were differentially expressed between the various s. aureus treatment groups.
these results demonstrate that different strains of s. aureus affect the proteome of the milk in different ways.
of specific interest was the unique response to the svc, heba  <dig> on day  <dig> pi.
therefore, investigating these changes in protein expression at later stages of infection is warranted.
overall, this study begins to shed light on the milk proteome and the changes that are elicited by intramammary infection with various strains of s. aureus and highlights an antimicrobial peptide, cpp <dig>  which may have novel therapeutic potential.

 mastitis, an inflammatory condition of the mammary gland, typically arises as a result of intramammary infections. in lactating dairy cattle, mastitis results in considerable monetary losses to the dairy industry due to depressed milk yield and quality, reduced reproductive performance, treatment, and pre-mature culling. staphylococcus aureus is a gram-positive, opportunistic, and contagious bacterium that is one of the most prevalent causes of mastitis in both humans and cattle. although infection can result in obvious clinical mastitis, s. aureus often evades the immune response resulting in a sub-clinical chronic infection that can persist for the life of the animal. classically, s. aureus is characterized as an extracellular pathogen. however, an increasing number of studies elude to its ability to invade and survive within various host cell types, including mammary epithelial cells, neutrophils, and macrophages; this may contribute to its persistence in various s. aureus-induced diseases. small colony variants of s. aureus may be particularly endowed for persistent survival by residing in host cells as they are characterized by slow-growth and atypical colony morphology, and are often over-looked by standard laboratory assays. it has been previously demonstrated that scvs can survive within professional and non-professional phagocytic cells, are more persistent than typical s. aureus strains, and are difficult to treat with conventional antibiotics. given that different s. aureus strains appear to utilize unique infection and survival strategies, we hypothesized that the host immune response will also vary in accordance with the infecting strain. to elucidate potential differences in host responses to s. aureus infection, the present study was carried out to investigate the kinetic response of key cytokines in serum and casein-depleted milk, as well as serum haptoglobin over a 21-day period following intramammary infection with three strains of s. aureus. additionally, a comparative proteomics platform was used to resolve the proteome of casein-depleted milk during acute and chronic phases of infection, and to identify differentially expressed proteins with potential immunomodulating effects. the s. aureus strains used for intramammary infection were: the scv heba a naturally occurring scv isolated from a dairy cow with history of chronic mastitis with atypical phenotypic properties, the parent strain displaying typical morphological and biochemical properties, and newbould, a teat skin strain that causes clinical bovine mastitis that is frequently used as a model for studying s. aureus-induced mastitis.  serum il- ifn-γ, tgf-β and haptoglobin response to s. aureus intramammary infection lactating dairy cattle were challenged with one of three s. aureus strains and the local and systemic host immune responses were characterized by monitoring cytokine kinetics and using a proteomics approach to identify proteins that may play a role in host defense. significant il- expression in the serum was not evident for any of the challenge groups until day post-infection. animals challenged with newbould and heba exhibited significantly higher il- expression on days and pi relative to levels on day. interestingly, animals challenged with parent showed a significant decrease in il- expression on day, and no significant change on day pi, relative to day significant differences in il- expression between the three challenge groups were observed on days and pi, with both newbould and heba challenge groups having significantly higher il- expression than the parent group. milk il- levels were not statistically significantly different from baseline levels at any time during the study. serum concentrations of ifn-γ were significantly up-regulated in the newbould challenge group during days 1- pi relative to serum ifn-γ levels at day. on day pi however, all challenge groups showed significant increases in ifn-γ expression compared to day. significant differences between challenge groups occurred on days 1- pi, whereby animals challenged with newbould had significantly higher serum ifn-γ levels compared to animals challenged with heba. serum tgf-β expression was significantly down-regulated in animals challenged with parent throughout days 1- and pi relative to day. in contrast, on day pi, the newbould and heba challenge groups had significantly up-regulated expression. on day pi, the heba challenge group was the only group to have significantly elevated expression of tgf-β compared to levels at day. significant differences in tgf-β expression between the three challenge groups were observed on day pi, with newbould and heba groups having significantly higher tgf-β levels compared to the parent group, and on day pi, with the heba group having significantly higher tgf-β expression compared to either newbould or parent groups. milk tgf-β levels were not statistically significantly different from baseline at any time during the study period. analysis of serum revealed significant elevations in haptoglobin concentrations within the first hours pi for all three challenge groups; however, linear and orthogonal polynomial contrasts did not reveal any significant differences in haptoglobin responses between any of the challenge groups. milk il- ifn-γ and tgf-β response to s. aureus intramammary infection significant changes in il- and tgf-β expression were not observed in casein-depleted milk for any of the challenge groups at any time, when compared to levels on day. in contrast, ifn-γ concentrations were significantly increased in animals challenged with s. aureus, but the response varied with the infection strain. for example, ifn-γ concentrations were increased in response to newbould on days 1- and day pi relative to levels on day, but not day pi. the parent challenge group also showed a significant increase in ifn-γ expression on days 1- pi. no significant change in ifn-γ expression was seen for the heba challenge group at any sampling time. significant differences between groups occurred on days 1- and day pi, where the newbould challenge group had higher ifn-γ expression compared to the heba group. milk proteome profiling following intramammary infection with s. aureus milk proteome analyses by 2-dimensional gel electrophoresis revealed protein spots that were differentially expressed on days or pi, relative to expression on day, with up-regulated expression occurring most frequently. in the newbould challenge group, there were and protein spots that were significantly induced on days and pi, respectively. in the group challenged with the parent strain, and protein spots were significantly induced on days and pi, respectively. lastly, for the heba challenge group, no proteins were induced on day pi, however, protein spots were significantly induced on day nb, newbould 305; p, parent 3231; heba, scv heba 3231 trmt*day denotes the treatment and day of the observed change in protein spot cpp component of the proteose peptone data are presented as the least square mean ± standard error of the mean. direction indicates the up-or down-regulation of the protein spot on day or pi, compared to baseline. spot i.d. number refers to protein spot number assigned by the melanie software. spots that were excised and identified by lc-ms/ms are indicated in parentheses. of the spots that were differentially expressed during the acute or chronic phases of infection, only spot was significantly differentially expressed over time and between the s. aureus challenge groups based on the fdr-adjusted p-value. this spot, and four other spots that were differentially expressed over time and between the treatment groups based on their raw p-values, were excised and subjected to protein sequencing by liquid chromatography and tandem mass spectrometry. identification of these protein spots and their expression patterns is summarized in table and figure three out of the spots were identified as component- of the proteose peptone. two of the spots corresponding to cpp were up-regulated in animals challenged with parent on day and pi, as well as in the heba challenge group on day pi, with treatment differences occurring on day pi between newbould and parent or heba challenge groups. in contrast, cpp was only up-regulated in the newbould challenge group on day pi, with significant treatment differences occurring between the newbould and heba challenge groups. aion score is = 10*log where p is the probability that the observed match is a random event;% coverage. btheoretical and observed masses and isoelectric points. discussion the current study aimed to monitor the host immune response to s. aureus intramammary infection using three unique strains, and to identify potential candidates for targeted therapies that may modulate the host defense to s. aureus challenge. intramammary infection with different strains of s. aureus resulted in variable cytokine responses within the bovine mammary gland and circulation, supporting our hypothesis that different strains of s. aureus prompt the activation of host immune responses that are unique to the infecting strain. typically, an increase in milk somatic cell count is indicative of an elevated and sustained inflammatory response caused by intramammary infection. during acute mastitis for example, neutrophil numbers in the mammary gland increase dramatically, constituting > 90% of milk somatic cells, whereas somatic cells in healthy glands contain < 22% neutrophils. cxcl-8/il- is the main tissue-derived chemoattractant for neutrophils; however, our observations demonstrated that the bovine il- response did not always parallel the somatic cell response during the different s. aureus intramammary infections. somatic cell scores were significantly elevated throughout the study period, and peaked during the acute phase in all challenge groups, however, we did not observe significant induction of il- in the casein-depleted milk at any point during the study period, and it was only detected in the serum on days and pi in animals challenged with newbould and heba the lack of an il- response in the milk is consistent with two previously reported independent studies carried out by bannerman et al. and riollet et al., suggesting that bovine il- is not the only chemokine involved in trafficking neutrophils to the infected mammary gland. it is possible that other chemoattractants, such as complement cleavage factor 5a, il-1β, tnf-α, cxcl and and granulocyte-macrophage colony stimulating factor produced by epithelial cells, macrophages and t cells, also contribute to neutrophil trafficking to the mammary gland during intramammary infection. for instance, neutrophil infiltration into the bovine mammary gland may be mediated by ifn-γ through the mechanism described by mcloughlin et al. that involves cxc chemokines other than il- such chemokines may include cxcl and cxcl which are constitutively expressed in bovine milk. in support of this, it has been demonstrated that il- and gm-csf are not active chemokines during lactation, whereas other factors such as il-1β and tnf-α are active during any stage of lactation. in the present study, ifn-γ was found to be highly expressed both locally, as indicated by levels in the milk, and systemically during days 1- pi in animals challenged with newbould which parallels previously reported results by bannerman et al.. serum ifn-γ levels were significantly up-regulated in all challenge groups at day pi compared to day this finding may be associated with the promotion of an enhanced cell-mediated immune response that includes neutrophil and macrophage activation. during the chronic phase of infection, differences in serum tgf-β expression between the three challenge groups were observed with notable increases being observed for the parent and heba groups. milk tgf-β did not significantly differ in response between any of the challenge groups; however, a trend towards altered tgf-β expression in animals challenged with newbould was observed, whereby the cytokine levels reached a maximum at hours pi. this observation is in line with a previous report of milk tgf-β kinetics upon newbould challenge in dairy cattle. the role of tgf-β in the host response to intramammary infection is uncertain because tgf-β is a pleiotropic immunoregulatory cytokine, with both pro- and anti-inflammatory functions, depending on the location and activation state of the cells that it is interacting with. this is exemplified by the cytokine's dual actions on macrophages. for instance, tgf-β suppresses tissue macrophage function and inflammation by down-regulating the production of chemokines and cytokines such as ifn-γ, up-regulating the expression of il- receptor antagonist, and suppressing the production of cytotoxic reactive oxygen and nitrogen intermediates. in contrast, tgf-β has also been shown to promote inflammation by inducing chemotaxis in human peripheral blood monocytes, enhancing their ability for transendothelial migration to sites of infection, and up-regulating the transcription of pro-inflammatory cytokines such as il-1β and tnf-α. furthermore, tgf-β has been shown to be an important factor for the differentiation of a subset of effector th cells known as th cells, which elicit inflammatory responses during certain pathogenic infections that are not sufficiently dealt with by th or th immunity. lastly, regulatory cells including tregs and m macrophages also produce tgf-β in humans. these cells help to prevent the deleterious effects of prolonged or excessive host immune responses, and this process includes suppressing various components of the immune response such as antigen presentation by antigen presenting cells, and promoting tissue repair by increasing the deposition of extracellular matrix at the site of tissue injury, and promoting angiogenesis. since tgf-β was not observed locally in the present study, it is unlikely that it was involved in repair of the mammary gland epithelium. there is evidence to suggest that s. aureus may be able to modulate the host immune response in part by promoting tgf-β expression; this could be a mechanism utilized by the heba strain for example, to promote its survival within the host. in support of this, staphylococcal superantigens have been demonstrated to induce the production of tgf-β by initiating the differentiation and expansion of tregs that down-regulate the inflammatory response. indeed, tgf-β has been implicated in playing a role in immunosuppression by up-regulating the expansion and/or activation of anergic regulatory t cells in the presence of s. aureus sags, which may disrupt class ii mhc antigen presentation by apcs, limiting cell-mediated immunity. interestingly, this immunomodulatory mechanism is also employed by leishmania as a means to suppress cell-mediated immunity and promote its intracellular survival. lastly, it should be noted that tgf-β expression may not necessarily denote its level of bioactivity, since a study by kehrl et al. demonstrated that human b cells largely express inactive tgf-β in response s. aureus cowan strain i. overall, haptoglobin concentrations increased with time in all challenge groups during the first days of the study period, and declined to initial levels by days and pi. however, no significant differences in serum haptoglobin levels were observed between the challenge groups. this may have been due to the chosen sampling times, as no samples were taken between days and pi. it has been previously demonstrated that haptoglobin is up-regulated during the acute phase response, and that it may be used to discriminate between acute and chronic inflammation in cattle. the findings in the present study indicate that overall serum haptoglobin levels were not significantly different between any of the challenge groups, suggesting that haptoglobin is a sensitive marker of inflammation due to s. aureus but it lacks specificity to discriminate the host response to the three different strains of s. aureus. it was previously demonstrated that two common mastitis-causing pathogens, escherichia coli and s. aureus, elicit different immune responses and pathogenesis. here, we demonstrate that the variability in immune protein response is also strain-specific, at least in the context of s. aureus. further studies investigating the profiles of other cytokines will add to the results of the current study. studying il- and il- for instance would provide insight into the involvement of th cells in s. aureus infections, as il- is a th cytokine and il- along with tgf-β supports the differentiation of this th subtype. furthermore, il- is one of the major signals for the induction of the acute phase response, and may therefore compliment the present haptoglobin data. monitoring chemoattractive factors, especially those under the regulation of tgf-β, would help delineate the pro- or anti-inflammatory role of tgf-β in intramammary infection with different strains of s. aureus. although it would have been interesting to measure il-1-β and tnf-α as well as other cytokines such as il- il- il- and il- that regulate the differentiation of these t-cell subpopulations, we had limited resources to carry out the cytokine analysis, and felt that inclusion of these cyctokies was beyond the scope of the current study as the aim was to note differences in host responses to different s. aureus strains, not to determine whether tgf-β is a pro- or anti-inflammatory cytokine. in addition to seeing unique cytokine profiles in response to the different strains of s. aureus, the present study also found a number of milk proteins to be differentially expressed at various time-points in response to infection, including host defense proteins. staphylococcus aureus insult to the mammary gland causes an inflammatory response that is characterized by an increase in vascular permeability, allowing exudates containing cells and proteins from the vasculature to enter the mammary gland. additionally, resident and recruited cells produce a number of proteins that target the pathogen and contribute to the restoration of homeostasis. therefore, the proteome of the milk may drastically change in response to pathogenic stimuli, and analysis of these changes may offer insight into the host response during mastitis. indeed, the present study demonstrated changes in the milk proteome during intramammary infection with s. aureus. in order to assess protein expression in milk during the acute and chronic phases of infection, samples were evaluated on days and pi, respectively. most proteins were up-regulated following newbould and parent challenges, especially on day pi during the acute phase of infection. on day pi, the number of up-regulated proteins was dramatically reduced in the newbould and parent challenge groups, as compared to day pi. results from this study indicate that differences in milk protein expression also occur as a result of intramammary infection with different strains of s. aureus. on day pi for example, challenges with newbould and parent induced milk proteins each, relative to day in contrast, significant changes in milk protein expression were not observed in response to challenge with heba at this time. on day pi however, milk proteins were induced by challenge with the parent strain and only protein was induced by newbould whereas proteins were significantly induced by the heba challenge. these results are indicative of the acute pathogenic nature of the parent and newbould strains and the unique phenotypic properties of scvs, which may elicit a milder host response. they suggest a highly active initial host response to challenges with the parent and newbould strains, then perhaps resolution or establishment of chronic infection by day pi. this corresponds with the somatic cell score data from these same cows presented by atalla et al., whereby the number of somatic cells in the milk during the chronic phase of infection still remained significantly up-regulated relative to day interestingly, we were not able to detect an initial host response to the scv strain, but the induction of protein expression was demonstrated on day pi. this delayed and relatively low level of host response corresponds to the ability of the heba strain to avoid recognition and persist within host cells. future studies should include later time points in order to determine if heba continues to up-regulate milk protein expression. two of the five spots corresponded to the most abundant milk proteins - β-casein precursor and β-lactoglobulin. this finding is likely a result of incomplete depletion of these highly abundant proteins. nonetheless, the depletion protocol was found to be sufficient to yield a comprehensive view of the milk proteome. of the five protein spots that were subjected to identification by lc-ms/ms, only one protein, cpp also known as lactophorin, was implicated in host defense. component of the proteose peptone is a phosphoglycoprotein uniquely expressed in the mammary gland of lactating dairy cattle and can be found in bovine milk whey. it has been previously suggested to have several functions that include antimicrobial activity, inhibition of lipolysis, mitogenic activity, and immunostimulation. it consists of seven polypeptide components, ranging in molecular weight from to kda. in the 2de gels, it occurred at three different locations ranging in mass from 15- kda and isoelectric point from 6-, which may correspond to its various components. in one study, the antimicrobial activity of cpp was investigated using a synthetic peptide called lactophoricin consisting of the 113- region of c-terminal of cpp. lactophoricin interacts with membrane phospholipids and forms voltage-dependent channels. thus, it has been hypothesized that it may also be involved in the pore forming of natural lipid bilayers such as bacterial membranes. indeed, campagna's results demonstrated that this peptide has inhibitory-growth effects on a number of gram-positive and gram-negative bacteria, with a more pronounced inhibitory effect on gram-positive bacteria, including s. aureus.  the present study demonstrates the complexity of the host immune protein response to various strains of s. aureus. intramammary infection with different strains of s. aureus, elicited unique host immune protein responses, as indicated by differential expression of the cytokines il- tgf-β, and ifn-γ in circulation and ifn-γ in casein-depleted milk. in general, cytokines displayed heightened responses in the serum compared to the casein-depleted milk. interestingly, the scv strain, heba showed increased levels of serum il- ifn-γ, and tgf-β occurring during the later stages of infection. whether this pattern of induction continues in later time points has yet to be determined. using a proteomic approach, we have also demonstrated that intramammary infection with three different strains of s. aureus elicits differential protein expression in bovine milk during the acute and chronic phases of infection. intramammary infection with newbould and parent strains resulted in pronounced induction of protein expression on day pi, while the heba strain elicited no protein induction at this time. on day pi, host protein activity was less pronounced for animals challenged with newbould and parent strains, while some induction occurred in animals challenged with heba lastly, cpp a potential antimicrobial peptide that is uniquely expressed in the bovine mammary gland during lactation, appeared on our 2de gels as three different spots, which were differentially expressed between the various s. aureus treatment groups. these results demonstrate that different strains of s. aureus affect the proteome of the milk in different ways. of specific interest was the unique response to the svc, heba on day pi. therefore, investigating these changes in protein expression at later stages of infection is warranted. overall, this study begins to shed light on the milk proteome and the changes that are elicited by intramammary infection with various strains of s. aureus and highlights an antimicrobial peptide, cpp which may have novel therapeutic potential. 
furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of s. aureus intramammary infection. notably, the protein, component- of the proteose peptone, was differentially expressed between the s. aureus treatment groups, implicating it as a potential antimicrobial peptide involved in host defense against s. aureus intramammary infection. changes in overall serum haptoglobin concentrations were observed for each s. aureus challenge group, but there were no significant differences observed between groups. intramammary infection of dairy cows with s. aureus is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. staphylococcus aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. therefore, it was hypothesized that the host immune response will be different for scv than its parental or typical strains of s. aureus. a sub-population of s. aureus, known as small colony variant, displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. in this study, the local and systemic immune protein responses to intramammary infection with three strains of s. aureus, including a naturally occurring bovine scv strain, were characterized. serum and casein-depleted milk cytokine levels, as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three s. aureus strains. serum il- ifn-γ, and tgf-β responses differed in dairy cows challenged with different strains of s. aureus. intramammary infection of dairy cattle with s. aureus causes an up-regulation of serum and milk immune-related proteins, and these responses vary depending on the infecting strain. proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection.
 staphylococcus aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. intramammary infection of dairy cows with s. aureus is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. a sub-population of s. aureus, known as small colony variant, displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. therefore, it was hypothesized that the host immune response will be different for scv than its parental or typical strains of s. aureus. in this study, the local and systemic immune protein responses to intramammary infection with three strains of s. aureus, including a naturally occurring bovine scv strain, were characterized. serum and casein-depleted milk cytokine levels, as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three s. aureus strains. furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of s. aureus intramammary infection.  serum il- ifn-γ, and tgf-β responses differed in dairy cows challenged with different strains of s. aureus. changes in overall serum haptoglobin concentrations were observed for each s. aureus challenge group, but there were no significant differences observed between groups. in casein-depleted milk, strain-specific differences in the host ifn-γ response were observed, but inducible il- and tgf-β concentrations were not different between groups. proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. notably, the protein, component- of the proteose peptone, was differentially expressed between the s. aureus treatment groups, implicating it as a potential antimicrobial peptide involved in host defense against s. aureus intramammary infection.  intramammary infection of dairy cattle with s. aureus causes an up-regulation of serum and milk immune-related proteins, and these responses vary depending on the infecting strain. 