Elsevier

Journal of Proteomics

Volume 221, 15 June 2020, 103763
Journal of Proteomics

Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: New perspectives for monitoring and understanding mastitis in dairy goats

https://doi.org/10.1016/j.jprot.2020.103763Get rights and content

Highlights

  • Differential shotgun proteomics of S. aureus-infected and healthy late lactation goat milk

  • The late lactation changes specifically associated to infection were identified

  • 38 DPs were unique to S. aureus infected milk, including haptoglobin

  • These may represent an alternative to the SCC for monitoring subclinical mastitis in late lactation

Abstract

The milk somatic cell count (SCC) is a standard parameter for monitoring intramammary infections (IMI) in dairy ruminants. In goats, however, the physiological increase in SCC occurring in late lactation heavily compromises its reliability. To identify and understand milk protein changes specifically related to IMI, we carried out a shotgun proteomics study comparing high SCC late lactation milk from goats with subclinical Staphylococcus aureus IMI and from healthy goats to low SCC mid-lactation milk from healthy goats. As a result, we detected 52 and 19 differential proteins (DPs) in S. aureus-infected and uninfected late lactation milk, respectively. Unexpectedly, one of the proteins higher in uninfected milk was serum amyloid A. On the other hand, 38 DPs were increased only in S. aureus-infected milk and included haptoglobin and numerous cytoskeletal proteins. Based on STRING analysis, the DPs unique to S. aureus infected milk were mainly involved in defense response, cytoskeleton organization, cell-to-cell, and cell-to-matrix interactions. Being tightly and specifically related to infectious/inflammatory processes, these proteins may hold promise as more reliable markers of IMI than SCC in late lactation goats.

Significance

The biological relevance of our results lies in the increased understanding of the changes specifically related to bacterial infection of the goat udder in late lactation. The DPs present only in S. aureus infected milk may find application as markers for improving the specificity of subclinical mastitis monitoring and detection in dairy goats in late lactation, when other widespread tools such as the SCC lose diagnostic value.

Introduction

Intramammary infections (IMI) and mastitis cause milk production losses and reduce dairy goat product quality. Subclinical mastitis due to chronic IMI can be especially problematic, and reliable monitoring and detection tools are needed for maintaining good profitability of goat productions [1]. The somatic cell count (SCC), that is, the number of cells per mL of milk, is largely considered a reliable IMI indicator in dairy ruminants [2]. In goats, however, the SCC is subjected to physiological variations related to age, parity, stage of lactation, estrus, and other factors [[3], [4], [5]], undermining specificity and limiting the diagnostic value of this practical and cost-effective marker. Late lactation, in particular, is associated with SCC increase in cow, sheep and goat milk [6], but the magnitude of this increase in goats is so high that SCC may not enable to distinguish infected from uninfected udders in late lactation [3,7,8]. Consequently, the reliability of the most widespread field tool, the California Mastitis Test (CMT), is severely affected [9]. The availability of a protein marker appearing in the milk only upon infection would increase the specificity of subclinical mastitis detection and support the screening of late lactation goats for IMI, enabling more meaningful management decisions especially at the dry-off [10,11].

The widespread adoption of milk SCC as an indicator of IMI is based on the notion that the number of cells in milk increases due to the active influx of neutrophils recalled into the milk as a result of the inflammation elicited by a microbial insult. Being this accompanied by increased permeability of the blood-milk barrier, with consequent leakage of serum contents into the milk, other ways to detect subclinical mastitis are based on these “leaked” proteins and other molecules found in the milk as a result of active secretion, cellular lysis or tissue rearrangements [11]. Investigating the proteome changes specifically associated with subclinical IMI is a suitable way to identify marker proteins that may represent a reliable alternative when the SCC loses specificity.

Gram-positive bacteria, and staphylococci in particular, are the most prevalent intramammary pathogens in dairy goats [[12], [13], [14], [15]]. Gram-positive bacteria cause mainly subclinical, chronic infections that persist along the dry period [6,8] justifying the need for more sensitive and specific screening tools for monitoring mammary gland health in dairy goats. Therefore, we selected Staphylococcus aureus subclinical IMI as the model condition for this study.

In summary, we applied a shotgun proteomics pipeline to compare late lactation, high SCC, S. aureus infected and uninfected milk with mid-lactation, low SCC uninfected milk to understand the changes induced by infection and to identify differential proteins with potential as subclinical mastitis markers in late lactation.

Section snippets

Animals and milk samples

Half-udder goat milk was retrieved from a frozen sample bank collected along the course of two entire lactations in a herd of Alpine goats farmed in Lombardy, Italy. All goats were clinically healthy for the two lactation years and showed no signs of mastitis. The detailed description of the herd and of experimental procedures was reported in a previous work [3]. Briefly, bacteriological analysis was carried out bi-monthly according to the National Mastitis Council standards [16] as described

Shotgun proteomics and differential analysis

S. aureus infected and uninfected milk was subjected to a shotgun proteomics workflow combining FASP, RP-HPLC, and high-resolution orbitrap MS. This led to the identification of 540 total unique proteins, of which 256 eligible for differential analysis. The complete description of the proteomic datasets is available in Pisanu et al. 2020 (Data in Brief, submitted).

To identify the changes specifically induced by S. aureus as opposed to the physiological changes occurring in late lactation, we

Discussion

Aim of this work was to detect and understand milk changes specifically related to IMI in dairy goats, especially focusing on late lactation, by differential label-free shotgun proteomics. This was also the first proteomic investigation of milk from goats with subclinical S. aureus mastitis.

When comparing the DPs observed in late lactation S. aureus infected and uninfected milk, the number, identity, and abundance indicated that the presence of S. aureus had a specific and strong impact on the

Acknowledgements

This work was funded by Sardegna Ricerche and the University of Milan.

Author contributions

Proteomic analysis and differential proteomics: SP, CC, DP. Microbiological analysis of milk: CP, MP. Animal examination, sample collection: VB. Funding and contribution to study design: SU, VB. Study conception, design and coordination, data analysis and interpretation, manuscript drafting: MFA. Data interpretation and manuscript revision: All authors.

Conflict of interest

The authors declare no conflict of interest.

Supplementary data

Supplementary data to this article can be found online.

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