Studies involving farm animals require the use of a large number of animals to obtain statistically meaningful data because these out-bred populations exhibit considerable genetic variation. Therefore, it is appealing to use intra-animal controls where possible, since this reduces genetic variation and fewer biological replicates are required. The quarters of the bovine mammary gland have been considered as separate physiological entities and therefore the use of one or more quarters as controls is a generally accepted experimental design [3–7, 11–16]. However, the results reported here demonstrate that the mammary gland quarters are interdependent during infection, with a profound change in the transcriptome of one quarter when neighbouring quarters are infected with S. aureus and especially E. coli. The control quarters remained bacteriologically negative throughout the study period  and therefore the observed differences are not due to contamination. The transcriptional response of the uninfected quarters was sufficiently high that it resulted in the masking of the response in infected quarters, when they were used as the base-line for DEG analysis (Table 1). In contrast, previous studies investigating the bovine mammary gland response to infection which have used within-animal controls have identified large numbers of differentially expressed genes [4–7]. However, in the earlier studies the animals were only challenged once, whilst in this study the animals were inoculated three times over 24 hours, which may have resulted in a greater signal triggering the response in the uninfected quarters.
Although a direct comparison between uninfected quarters from healthy and uninfected animals was not made in this study, the effect of infection in neighbouring quarters can be inferred from the differences observed between the various control samples. Recently, a microarray experiment comparing the transcriptome of uninfected quarters from healthy and E. coli infected animals was reported . A total of 476 DEG were identified, confirming that E. coli infection alters the transcriptional response of neighbouring mammary gland quarters. Over twenty eight percent (53) of the DEG identified in our comparison of E. coli and S. aureus control quarters were also found in this study, which utilized the bovine Affymetrix platform, including ABCG2 and FKBP5. The direction of the transcriptional change observed in these 53 genes was the same in both studies, i.e. those up-regulated during E. coli infection of neighbouring quarters  were found at higher levels in EC24T0 samples than SA24T0 samples, suggesting that the differential expression of at least these 53 genes is associated with E. coli infection and not S. aureus infection. The broader intensity plots of the EC24T0 samples illustrated that there was a greater range of transcriptional change in these samples compared to the reference than observed in the SAT0 samples. This may suggest that the vast majority of the differential expression is due to the E. coli infection of neighbouring quarters. Furthermore, the transcriptome of the uninfected quarters was indistinguishable from that of quarters infected with E. coli for six hours.
However, the design of the experiment reported here does not allow us to conclusively say that the transcriptional differences are all due to changes in expression in the E. coli control quarters. Indeed, the comparison of control quarters from animals infected with S. aureus for 24 and 72 hours has revealed that S. aureus also induces transcriptional changes in neighbouring quarters 24 hours post infection, which may account for some of the differences observed between SA24T0 and EC24T0. Furthermore, XDH, which is involved in the generation of the bactericidal agent peroxynitrate and is believed to play an important role in the mammary gland immune response (reviewed by ), is expressed at significantly higher levels in SA24T0 than EC24T0 and therefore may be induced during S. aureus infection of neighbouring quarters. Moreover, additional evidence that S. aureus infection induces a response in neighbouring quarters has recently been reported. The expression of the acute phase proteins serum amyloid A3 (SAA3) and haptoglobin (HP) as well as the β-defensin DEFB5 was elevated in uninfected control quarters from animals infected with a low virulence strain of S. aureus compared to tissue from healthy animals .
The initial statistical analysis of the microarray data comparing the S. aureus control quarters from the different time courses failed to detect any significantly differentially expressed genes. Although this could be a genuine result, S. aureus infection generally induces a milder immune response than E. coli, modulating gene expression to a lesser extent at the site of infection, which would impact on the signals being detected by the neighbouring quarters. This variation, compounded with the well-known fact that individual cattle differ greatly in their response to udder infections (reviewed by ), would result in considerable variability in the response of neighbouring quarters to S. aureus infection and could account for the lack of statistical significance, which would be further exacerbated by the low number of biological replicates included in this study. Indeed, lowering the stringency of the microarray analysis allowed the identification of over 70 genes, including several genes known to be associated with the mammary gland response to infection, e.g. SOD2, IL1B, LTF, S100A8 and S100A12. Nearly one sixth of the differentially expressed genes are associated with the response of cells to stress. Furthermore, IPA revealed that a network of IL1 and associated molecules were expressed at higher levels in SA24T0 samples than in the SA72T0 samples, which are a well-known trigger for inflammation. Paradoxically, the cattle infected with S. aureus did not show signs of inflammation, even in the infused udder quarters, 24 hours post infection , when the elevated levels of IL1 and related molecules were detected in this study. Conversely, at 72 hours, when the IL1 response was lower in uninfected quarters, the infected quarters showed up-regulation of inflammation-associated genes . Therefore, S. aureus infection may initially elicit a slight systemic inflammatory response that induces IL1 expression in uninfected quarters, which disappears during sustained infection. It is unclear if S. aureus actively contributes to the down-regulation of the systemic response, which may contribute to the typically sub-clinical disease pattern observed in S. aureus mastitis.
Some of the transcriptional changes may relate to changes in the cellular composition of the mammary gland. Bacterial infections induce the influx of immune cells, predominantly neutrophils, into the infected mammary gland quarter, which leads to increasing SCC in milk. The SCC measured in the control quarters from animals included in this study increased during the time course of both E. coli and S. aureus infection . This is in agreement with previous studies which found significantly higher SCC in milk from uninfected quarters of naturally infected animals compared to those from healthy animals [25–27]. Moreover, the percentage of neutrophils was significantly higher, whilst the percentage of macrophages was significantly lower, in milk from uninfected quarters from animals with mastitis compared to those from healthy animals . Differential expression of cell surface markers, e.g. CD74, Fc receptors and CD4, are indicative of differences in the cellular composition of the control quarters from E. coli and S. aureus infected animals. Levels of CD4+ T lymphocytes increase in milk during S. aureus infection [26, 28] and the results reported here suggest that some cells may originate from the uninfected quarters.
The influx of immune cells may not account for all the transcriptional changes of uninfected quarters. The response of the first quarter to infection with E. coli was significantly different from that of the second infected quarter after the same period of infection. The profound increase in SCC observed in milk from the first quarter 12 hours post infection was not observed in milk samples from the second quarter infected 12 hours later . The earlier infection appears to have primed the neighbouring mammary gland quarters to respond differently to infection. Similar results have been reported recently in an experimental infection study where one animal showed signs of infection in one udder quarter before the experiment started . This animal was still included in the study, using the other, uninfected udder quarters. The proinflammatory cytokine response, i.e. up-regulation of IL1B, IL6, IL8 and tumour necrosis factor (TNF), of this animal was more pronounced than that observed in other animals included in the study . The priming of the mammary gland may persist after infection. Experimental E. coli infection of individual quarters fourteen days after infection of neighbouring quarters resulted in significantly less bacteria being isolated from the milk than observed with the initial infection . Furthermore, it was recently reported that priming udders with a low dose of E. coli lipopolysaccharide (LPS) protects against experimental infection with E. coli pathogens . Moreover, LPS priming induces sustained expression of bactericidal factors (e.g. β-defensins) by bovine mammary epithelial cells (MEC), whilst reducing the over expression of pro-inflammatory cytokines which may be harmful to the animal . However, there is little overlap in DEG from primed MEC and those observed in the uninfected quarters included in this study, suggesting that different mechanisms may be involved in priming neighbouring quarters.
TGFB1 levels were observed to be much lower in the control quarters from E. coli infected animals than those from animals infected with S. aureus. Therefore, it is interesting to hypothesize that the priming of the udder quarters involves the suppression of levels of TGFB1 and associated molecules which was revealed by IPA. Moreover, the same pattern of TGFB3 expression has previously been reported using the same experimental samples . The TGFBs are pleiotropic cytokines which play an important role in multiple biological processes including regulating the adaptive and innate immune responses. TGFB1 inhibits inflammation by, amongst other mechanisms, suppressing the activation of several immune cells, e.g. NK cells and macrophages, and although chemotactic, can inhibit neutrophil transmigration (reviewed by ). Further analysis has shown that significantly lower levels of TGFB1 mRNA is present in the uninfected quarters of E. coli infected animals included in this study than in the quarters of healthy animals involved in another study  (Additional file 3). It appears that animals down regulate the expression of TGFB1 in the udder when a neighbouring quarter has been challenged with E. coli pathogens. This reduction in the level of the immunosuppressor TGFB1 would lower the threshold for triggering the induction of a variety of immunologically relevant genes, resulting in a faster and more robust response to infection. In contrast, the weaker stimulus elicited by invading S. aureus pathogens maintains, or possibly enhances (Additional file 3), the relatively high levels of TGFB1 present in uninfected quarters, thereby buffering immune stimuli in the healthy, uninfected udder quarter.
Response to Stress was one of the major GO Biological Processes categories identified in both the comparisons of EC24T0 and SA24T0 and that of S. aureus control quarters. Many of the genes within this category have previously been identified as being involved in the immune response to mastitis, including LBP, STAT3, S100A8 and SOD2. However, many of the most differentially regulated genes, e.g. ABCG2 and FKBP5, have not, to our knowledge, previously been associated with mastitis. Both ABCG2 and FKBP5 were also identified as DEG in the recent transcriptional comparison of uninfected quarters from healthy and mastitic animals , suggesting that their differential expression was due to the infection of neighbouring quarters with E. coli. Furthermore, the expression of both genes was modulated in E. coli infected mammary gland quarters .
ABCG2, also known as breast cancer resistance gene (BCRP), is a xenobiotic transporter that is expressed at high levels in lactating mammary glands of cows, humans and mice [34, 35]. ABCG2 plays an important role in the transport of nutrients into milk, especially riboflavin (vitamin B2)  and has been identified as the candidate gene underlying a quantitative trait locus on bovine chromosome 6 that affects milk yield and composition [37, 38]. Therefore, the down-regulation of ABCG2 in quarters neighbouring E. coli infected quarters may be associated with the decrease in milk yield and casein synthesis, which was observed in these animals [10, 39] and may imply a nutrient deficiency in milk from these animals.
The immunophilin FKBP5 has several functions, including mediating the action of the immunosuppressive drug FK506. The FKBP5-FK506 complex binds protein phosphatase 3, catalytic subunit, alpha isozyme (PPP3CA), also known as calcineurin, thus preventing it from phosphorylating and activating NFAT transcription factors, which in turn prevents T cell activation and proliferation . FKBP5 is highly expressed in murine T cells  and therefore the up-regulation observed during E. coli infection may be due to lymphocyte migration into mammary gland tissue. In addition, FKBP5 is induced by glucocorticoids and is believed to be involved in the negative feedback loop for glucocorticoid receptor signalling . FKBP5, along with heat shock protein 90 (HSP90), is a component of the glucocortcoid receptor complex. The HSP90 family member HSP90AB1 exhibits a similar expression profile as FKBP5 in both this study and Mitterheumer et al. . Glucocorticoids are able to modulate NF-κB activity by a FKBP5-dependent pathway . FKBP5 interacts with conserved helix-loop-helix ubiquitous kinase (IKKα), which phosphorylates nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), the inhibitor of NF-κB, resulting in IκBα proteosome-mediated degradation and the nuclear translocation and activation of NF-κB .
The mechanism by which the infection of neighbouring quarters affects the transcriptome of uninfected quarters is unknown. Mastitis induces a systemic effect, inducing transcriptional changes in the liver , including the production of acute phase proteins which are released into the blood that circulates around all the udder quarters. This has been postulated to explain the transcriptional profile in healthy quarters of animals infected with E. coli. Furthermore, it has become clear that endogenous damage or danger signals  also lead to inflammation  and evidence now supports their role in the response to pathogens . Therefore, it is possible that the uninfected udder quarters are responding to such danger or damage signals released into the extracellular environment following infection in the neighbouring quarters. However, analysis of TLR and β-defensin expression in the local and peripheral lymph nodes of animals included in this study revealed that a response was only observed in the lymph nodes draining the quarters infected with E. coli for 12 and 24 hours , suggesting that the systemic effect is quite limited. Local cross-talk between the udder quarters must, therefore, be responsible for priming the neighbouring udder quarters. Indeed, lymphocytes have been shown to be capable of migrating between mammary gland quarters  and therefore the presence of a more localized interaction between quarters is probable and requires further investigation.