Prednisolone-induced predisposition to femoral head separation and the accompanying plasma protein changes in chickens

Abstract

Femoral head separation (FHS) is an idiopathic bone problem that causes lameness and production losses in commercial poultry. In a model of prednisolone-induced susceptibility to FHS, the changes in plasma proteins and peptides were analyzed to find possible biomarkers. Plasma samples from control and FHS-susceptible birds were depleted of their high abundance proteins by acetonitrile precipitation and were then subjected to cation exchange and reverse-phase (RP) fractionations. Analysis with matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) showed several differentially expressed peptides, two of which were isolated by RP-HPLC and identified as the fragments of apolipoprotein A-I. The acetonitrile fractionated plasma proteins were subjected to reduction/alkylation and trypsin digestion followed by liquid chromatography and tandem mass spectrometry, which showed the absence of protocadherin 15, vascular endothelial growth factor-C, and certain transcription and ubiquitin-mediated proteolytic factors in FHS-prone birds. It appears that prednisolone-induced dyslipidemia, vascular, and tissue adhesion problems may be consequential to FHS. Validity of these biomarkers in our model and the natural disease must be verified in future using traditional approaches.

Biomarker insights: Lameness because of femoral head separation (FHS) is a production and welfare problem in the poultry industry. Selection against FHS requires identification of the birds with subclinical disease with biomarkers from a source such as blood. Prednisolone can induce femoral head problems and predisposition to FHS. Using this experimental model, we analyzed the plasma peptides and proteins from normal and FHS-prone chickens by mass spectrometry to identify differentially expressed peptides and proteins. We found two peptides, both derived from apolipoprotein A-I, quantitatively elevated and two proteins, protocadherin 15 and VEGF-C, that were conspicuously absent in FHS-susceptible birds.

Keywords: biomarker; chickens; femoral head separation; glucocorticoids; mass spectrometry; proteomics.

Figure 1

Histology showing prednisolone-induced bone marrow adipogenesis (arrows indicate adipocytes).

Figure 2

(A) Comparison of MADLI-TOF mass spectra of C18 fractionated HAPD plasma showing the m/z 7304 peak analyzed by ClinproTools. The average spectra from CTRL (red) and FHS (green) groups represent the cumulative results of three pooled samples from nine birds. (B) Peptide mass fingerprint (PMF) of 7304 Da peptide. (C) Tandem mass spectrometry (MS/MS) of 1120 Da tryptic fragment derived from 7304 Da peptide.

Figure 3

(A) Comparison of MADLI-TOF mass spectra of SCX fractionated HAPD plasma showing m/z 3203 region analyzed by ClinproTools. The average spectra from CTRL (red) and FHS (green) groups represent the cumulative results of three pooled samples from nine birds. (B) Peptide mass fingerprint (PMF) of 3203 Da peptide. (C) Tandem mass spectrometry (MS/MS) of 1318 Da tryptic fragment derived from 3203 Da peptide.

Figure 4

(A) Protein sequence of chicken apolipoprotein A-I and the corresponding regions representing the 7304 Da (underlined) and the 3203 Da peptide (shown in bold) and (B) the possible cleavage sites that may generate these peptides, predicted by PROSPER online software.