Towards Understanding Non-Infectious Growth-Rate Retardation in Growing Pigs

Abstract

For growth-rate retardation in commercial growing pigs suffering from non-infectious diseases, no biomarker is available for early detection and prevention of the condition or for the diagnosis of affected animals. The point in question is that the underlying pathological pathway of the condition is still unknown and multiple nutritional or management issues could be the cause of the disease. Common health status markers such as acute phase proteins, adenosine deaminase activity or total antioxidant capacity did not show any alteration in the saliva of animals with growth-rate retardation, so other pathways should be affected. The present study investigates saliva samples from animals with the same commercial crossbreed, sex and age, comparing control pigs and pigs with growth-rate retardation. A proteomics approach based on two-dimensional gel electrophoresis including mass spectrometry together with validation experiments was applied for the search of proteins that could help understand disease mechanisms and be used for early disease detection. Two proteins were detected as possible markers of growth-rate retardation, specifically S100A12 and carbonic anhydrase VI. A decrease in innate immune response was confirmed in pigs with growth-rate retardation, however further studies should be necessary to understand the role of the different CA VI proteoforms observed.

Keywords: biomarker detection; gel-based proteomics; growth-rate retardation; pig; saliva.

Figure 1

Gel images of porcine saliva samples from a pig with growth-rate retardation (a) and a control pig (b). Red marked spots show statistically significant changes in abundance between the two health status conditions. For details on spot changes and protein identifications see Table 3, Table 4 and Table 5, respectively.

Figure 2

Close-up of the 2-DE region of pH 7–9 and MW 45–30 kDa for a pig with growth-rate retardation (a) and a control pig (b). Red marked spots (54–62) correspond to carbonic anhydrase VI protein. Fold change of carbonic anhydrase VI spots chains. Fold change: ratio mean value in GRR/mean value in control pigs. Mean value: percentage of spot volume.

Figure 3

SDS-PAGE image of a group of 10 control pigs (lanes 2–6 of each gel) and a group of 10 pigs with growth-rate retardation (lanes number 7–11 of each gel). Lanes 1 show molecular weight markers in kDa. Red arrows show statistically significant changes in band abundance between the two groups of animals that were subjected to MS analysis for protein identification. For details of band changes and protein identifications see Table 5 and Supplementary Material Table S1, respectively.

Figure 4

Signal detection after western blot analysis against S100A12 protein in control pigs (lane number 1–10) and pigs with growth-rate retardation (lane numbers 11–27; lane numbers 11–20 represent GRR pigs used in the proteome approach while lane numbers 21–27 show results of an additional group of 7 GRR pigs from the same farm) (a). Levels of the integrated signal intensity observed in control pigs and pigs with growth-rate retardation (Pigs with GRR I n = 10 and Pigs with GRR II n = 17) (b). Data were normalized using an internal control sample. The plot shows median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) and outliers (•). Asterisk represents the statistically significant differences between groups of pigs. * p < 0.05. To see the whole WB image and the membrane staining after WB analysis see Supplementary Material Figure S2.