Quantitative milk proteomics – Host responses to lipopolysaccharide‐mediated inflammation of bovine mammary gland

Abstract

Intramammary infusion of lipopolysaccharide (LPS) in cows induces udder inflammation that partly simulates mastitis caused by infection with Gram-negative bacteria. We have used this animal model to characterize the quantitiative response in the milk proteome during the time course before and immediately after the LPS challenge. Milk samples from three healthy cows collected 3 h before the LPS challenge were compared with milk samples collected 4 and 7 h after the LPS challenge, making it possible to describe the inflammatory response of individual cows. Quantitative protein profiles were obtained for 80 milk proteins, of which 49 profiles changed significantly for the three cows during LPS challenge. New information obtained in this study includes the quantified increase of apolipoproteins and other anti-inflammatory proteins in milk, which are important for the cow's ability to balance the immune response, and the upregulation of both complement C3 and C4 indicates that more than one complement pathway could be activated during LPS-induced mastitis. In the future, this analytical approach may provide valuable information about the differences in the ability of individual cows to resist and recover from mastitis.

Veterinary Biomarker Discovery: Proteomic Analysis of Acute Phase Proteins

“…The prospect of using APPs as biomarkers of inflammation and infection for veterinary applications has inspired a significant body of research, including the development of antibodies and other quantitative methods for analyzing APP expression during disease in food animal species (Murata et al, 2004). More recently, interest in APPs as potential veterinary biomarkers has led to the application of proteomic strategies for the evaluation of APPs and measures of the host response in complex biological samples (Bendixen et al, 2011;Boehmer et al, 2010;Danielsen et al, 2010;Smolenski et al, 2007).…”

“…The first attempt to characterize proteins related to host defense in mastitic bovine milk did result in the identification of the APPs serum albumin, fibrinogen, and SAA, but very few biological replicates were used in the analyses, and the objectives were strictly proteome coverage, not comparisons between the healthy and diseased states (Smolenski et al, 2007). The two most recent comparative proteomic analyses of normal versus mastitic bovine milk, however, have not only identified several APPs in milk, but tracked changes in APPs over the course of infection, and quantified modulation in relative abundance of APPs during disease (Boehmer et al, 2010;Danielsen et al, 2010 (Mitchell et al, 2008;2007) Table 2. Acute phase proteins identified disease in food animals using proteomic strategies…”

“…Proteomics has also been applied to the study of the gastrointestinal tract in swine (Wang et al, 2009;Danielsen et al, 2007Danielsen et al, , 2006, as well as dietinduced fatty liver disease in Ossabaw pigs . Studies aimed at the elucidation of potential biomarkers of bovine mastitis have specifically dominated veterinary biomarker initiatives in cattle, however, due in large part to affiliated inflammation, economic and food safety concerns, and the lack of efficacious treatment options (Boehmer et al, 2010;Danielsen et al, 2010;Smolenski et al, 2007;Hogarth et al, 2004). Several comparative proteomic studies have focused on the identification of diagnostic biomarkers of mastitis in bovine milk, though the proteomic analysis of mammary tissue from healthy cows and cows with clinical mastitis has also been conducted (Yang et al, 2009).…”

“…Similar to the 2D-GE-MALDI-TOF/MS of bovine milk, the proteomic analysis of changes in the bovine BALF proteome induced by dexamethasone revealed increases in the APPs alpha-2-HS-glycoprotein, alpha-1-antichymotrypsin, alpha-1-antitrypsin, and AGP in treated animals when compared to controls (Mitchell et al, 2007). To avoid some of the limitations imposed by a 2D-GE-MALDI-TOF/MS proteomic experiment, more recent proteomic analyses o f b o v i n e m i l k h a v e b e e n a c c o m p l i s h e d through the use of LC-MS/MS (Boehmer et al, 2010;Danielsen et al, 2010;Smolenski et al, 2007). The first attempt to characterize proteins related to host defense in mastitic bovine milk did result in the identification of the APPs serum albumin, fibrinogen, and SAA, but very few biological replicates were used in the analyses, and the objectives were strictly proteome coverage, not comparisons between the healthy and diseased states (Smolenski et al, 2007).…”

“…Accordingly, recent veterinary biomarker discovery initiatives have focused on the identification of sensitive and reliable indicators for use in evaluating the efficacy of adjunctive therapies for the treatment of inflammation associated with disease in food animal species. As a result, modulation in several APP, including haptoglobin (HPT), alpha-1-acid glycoprotein (AGP), bovine serum albumin (BSA), inter-alpha-trypsin inhibitor heavy chain-4 (ITIH4), and serum amyloid A (SAA), have been detected in the analyses of bovine milk, plasma, and broncheoalveolar lavage fluid (BALF) during both naturally occurring and experimentally induced disease (Boehmer et al, 2010;Danielsen et al, 2010;Mitchell et al, 2007;Eckersall et al, 2006). Classic approaches to the characterization of protein changes in complex biological fluids have required the availability of species-specific antibodies for the detection and quantification of a given protein.…”