Research paperDetection of potential markers for systemic disease in saliva of pigs by proteomics: A pilot study
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.
Section snippets
Animals and sampling procedure
Twenty finishing pigs, conventional Duroc × (Landrace × Large White), from a farm located in the northwest of Murcia, Spain, were used in the present study. All animals were non-castrated males from the same age and housing conditions in order to have as little variation as possible except for the health state. Animals came from a commercial finishing unit of 1800 animals and received a nutritionally balanced commercial diet and water ad libitum. Pigs were vaccinated twice against Mycoplasma
Health status characterization of animals
Though the ante-mortem inspection performed at farm during the veterinary visit, all pigs included in group 1 had no clinical sings of disease. On the other hand, animals from group 2 presented several different symptoms of disease: 4 out of 10 had a cough, one pig showed several abscesses in legs and abdomen, external superficial injuries were observed in 5 pigs and arthritis appeared in 3 animals. Moreover, growth retardation was observed in 9 out of 10 animals.
Animals from group 1 presented
Discussion
Porcine saliva is obtained by non-invasive and minimally stressful sampling methodologies. Its analysis has proved to be very useful in porcine production for monitoring health status (Gutiérrez et al., 2009) and animal welfare (Fuentes et al., 2011). While previous studies in our laboratory have characterized the salivary proteome map of healthy pigs (Gutiérrez et al., 2011), no studies have compared protein patterns of pigs with different health status. A comprehensive evaluation of proteins
Conclusion
Overall, the salivary protein profiles, obtained in healthy and diseased animals display the ability of whole saliva to reflect systemic conditions. This implicates that, after a necessary protein analysis validation, the present results would establish salivary biomarkers which may be of help for monitoring herd health status at farm level or for detecting individual diseased animals at the slaughterhouse.
Acknowledgements
This study has been possible thanks to Integrated Action (HA-2008-0016) between the Department of Animal Medicine and Surgery of The University of Murcia, Spain and the Department of Biomedical Sciences, The University of Veterinary Medicine, Vienna, Austria.
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