Detection of potential markers for systemic disease in saliva of pigs by proteomics: A pilot study

Abstract

Animals with different health status have been studied in order to extend the knowledge about protein composition of porcine saliva samples and to discover potential salivary markers for systemic disease in porcine production. Clinical examination of animals was performed at farm level where 10 healthy pigs and 10 animals with evident clinical signs of disease were randomly selected. Saliva and blood samples were obtained and afterwards animals were humanely sacrificed to perform a complete necropsy. Levels of two acute phase proteins, haptoglobin and C-reactive protein, were used to identify possible active infections of the animals. Moreover, serological analysis, to the main porcine infectious diseases in the area, was performed. Salivary proteins were separated by two-dimensional gel electrophoresis followed by mass spectrometry for the identification of specific proteins. A total of 58 spots out of 75 were successfully identified by MS, which correspond to 20 unique proteins. Two different approaches were used to perform a statistical comparison of saliva protein patterns from healthy and diseased animals using the relative spot volume (% spot volume/total volume of all spot in the gel, approach “A”) or taking also into account the total protein content of each saliva sample (μg of spot/mL of saliva, approach “B”). Both analyses showed three proteins in common that are differentially regulated between states. However, approach B was selected for biomarker searching since it gave an estimation of protein concentration and showed differential expression of proteins between both health states in a total of 10 proteins, which were up-regulated in disease. Mass spectrometric analysis identified those proteins as salivary lipocalin, lipocalin 1, double headed protease inhibitor protein, adenosine deaminase, haptoglobin, albumin fragments, S100-A8, S100-A9, S100-A12 and pancreatic alpha amylase. These proteins could be considered as potential salivary markers of disease.

Introduction

The identification of biomarkers of specific pathologic processes is very useful for the early detection of disease in farm animals (Castronovo et al., 2007). Proteomics has become a valuable laboratory tool for determining protein expression patterns and it has been widely used for new biomarker discovery in complex biological samples (Shin et al., 2006, Hennig-Pauka et al., 2006).

Saliva is a complex body fluid composed by a mixture of secretions of multiple salivary glands and by numerous minor glands in the lip, cheek, tongue and palate, containing also serum products, nasal and bronchial secretions, epithelial cells and microbes or their products (Sondej et al., 2009). Saliva is nowadays becoming more and more interesting as a clinical tool because of its potential to reflect both oral and systemic health conditions (Baldini et al., 2008). In human medicine a wide number of studies have sought to develop a complete catalogue of proteins in saliva that are relevant not only for basic research but also for its use as a diagnostic tool (Messana et al., 2008). Moreover, several disease-specific biomarkers have been reported in human saliva proteomic analysis, for instance for breast cancer (Streckfus and Bigler, 2005), head and neck squamous cell carcinoma (Dowling et al., 2008), Sjogren's syndrome (Giusti et al., 2007), and diabetes type 2 (Rao et al., 2009) diagnosis.

The proteome maps of several porcine organs, cells and body fluids have been described, such as for serum (Miller et al., 2009), hepatocytes (Caperna et al., 2008), platelets (Esteso et al., 2008), peripheral blood mononuclear cells (Ramirez-Boo et al., 2006), skeletal muscle (Kim et al., 2004), seminal plasma (Strzezek et al., 2005), and testis (Huang et al., 2005). Furthermore, proteomics has revealed the identification of a number of differentially expressed proteins related to tenderness in muscle samples (Lametsch et al., 2003) and also after an infection with Actinobacillus pleuropneumoniae in bronchoalveolar lavage fluid (Hennig-Pauka et al., 2006).

In a recently reported proteomic analysis, we have characterized the proteome map of porcine saliva in a healthy state (Gutiérrez et al., 2011), since a crucial first step in biomarker discovery in systemic disease is the characterization of the normal saliva proteome (Hu et al., 2005). The aim of the present study was to further expand our previous saliva proteome map and to analyze the proteins differentially expressed in saliva samples of healthy control animals and pigs with evident clinical signs of disease. The study was carried out by using two-dimensional gel electrophoresis (2-DE), to separate the protein components, followed by mass spectrometry (MS), to identify the peptides produced by in-gel digestion of the spots of interest.