Escherichia coli- and Staphylococcus aureus-induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters

Abstract

Background: The most important disease of dairy cattle is mastitis, caused by the infection of the mammary gland by various micro-organisms. Although the transcriptional response of bovine mammary gland cells to in vitro infection has been studied, the interplay and consequences of these responses in the in vivo environment of the mammary gland are less clear. Previously mammary gland quarters were considered to be unaffected by events occurring in neighbouring quarters. More recently infection of individual quarters with mastitis causing pathogens, especially Escherichia coli, has been shown to influence the physiology of neighbouring uninfected quarters. Therefore, the transcriptional responses of uninfected mammary gland quarters adjacent to quarters infected with two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were compared.

Results: The bacteriologically sterile, within-animal control quarters exhibited a transcriptional response to the infection of neighbouring quarters. The greatest response was associated with E. coli infection, while a weaker, yet significant, response occurred during S. aureus infection. The transcriptional responses of these uninfected quarters included the enhanced expression of many genes previously associated with mammary gland infections. Comparison of the transcriptional response of uninfected quarters to S. aureus and E. coli infection identified 187 differentially expressed genes, which were particularly associated with cellular responses, e.g. response to stress. The most affected network identified by Ingenuity Pathway analysis has the immunosuppressor transforming growth factor beta 1 (TGFB1) at its hub and largely consists of genes more highly expressed in control quarters from S. aureus infected cows.

Conclusions: Uninfected mammary gland quarters reacted to the infection of neighbouring quarters and the responses were dependent on pathogen type. Therefore, bovine udder quarters exhibit interdependence and should not be considered as separate functional entities. This suggests that mastitis pathogens not only interact directly with host mammary cells, but also influence discrete sites some distance away, which will affect their response to the subsequent spread of the infection. Understanding the underlying mechanisms may provide further clues for ways to control mammary gland infections. These results also have implications for the design of experimental studies investigating immune regulatory mechanisms in the bovine mammary gland.

Figure 1

Differential expression in uninfected udder quarters from animals infected with Escherichia coli and Staphylococcus aureus. Preliminary analysis of the microarray data revealed different patterns of gene expression in control quarters from animals infected with Escherichia coli and Staphylococcus aureus. The log normalized intensity values (sample [Cy5]/reference [Cy3]) were plotted for the combined data from control quarters from animals infected with E. coli (EC24T0) and S. aureus (SAT0) using GeneSpring (Agilent). Both plots have a normal-type distribution around 1. EC24T0 have a broader peak of intensity values suggesting a greater range of expression compared to SAT0.

Figure 2

TGFB1 Nework. Ingenuity pathway analysis of the EC24T0 v SA24T0 DEG list identified an interaction network involving TGFB1 and associated molecules with a network score of 38. Green denotes molecules that were expressed at higher levels in SA24T0 samples and red denotes those more highly expressed in EC24T0 samples.

Figure 3

Validation of EC24T0 v SA24T0 differentially expressed genes. Summary of the quantitative RT-PCR (RT-qPCR) results for ten selected genes that were identified from the microarray data analysis as exhibiting differential expression between the control quarters from E. coli (EC24T0) and S. aureus (SA24T0) infected cattle. The results are represented as the average mRNA level detected in EC24T0 (grey bars) and SA24T0 (white bars) relative to the sample with the lowest expression. The bars denote standard error of the mean, *, ** and *** denote that the expression difference is statistically significant with P < 0.05, P < 0.005 and P < 0.0005 respectively.

Figure 4

IL1 Network. Ingenuity pathway analysis of the SA24T0 v SA72T0 DEG list identified an interaction network involving the pro-inflammatory cytokine IL1 with a network score of 42. Red denotes molecules that were expressed at higher levels in SA24T0 samples and green denotes those more highly expressed in SA72T0 samples.

Figure 5

Validation of SA24T0 v SA72T0 differentially expressed genes. Summary of the quantitative RT-PCR (RT-qPCR) results for selected genes between the control quarters of animals infected with S. aureus for 24 hours (SA24T0) or 72 hours (SA72T0). The results are represented as the average mRNA level detected in SA24T0 (white bars) and SA72T0 (grey bars) relative to the sample with the lowest expression. The bars denote standard error of the mean, * and ** denote that the expression difference is statistically significant with P < 0.05 and P < 0.005 respectively. A. RT-qPCR results for six selected genes that were identified from the microarray data analysis as exhibiting differential expression between SA24T0 and SA72T0. B. RT-qPCR results comparing the differential expression of ABCG2, CXCL12, IDH1, TGFB1 and XDH between SA24T0 and SA72T0 samples.