Ovine serum biomarkers of early and late phase scrapie

Abstract

Background: Transmissible spongiform encephalopathies are fatal neurodegenerative disease occurring in animals and humans for which no ante-mortem diagnostic test in biological fluids is available. In such pathologies, detection of the pathological form of the prion protein (i.e., the causative factor) in blood is difficult and therefore identification of new biomarkers implicated in the pathway of prion infection is relevant.

Methods: In this study we used the SELDI-TOF MS technology to analyze a large number of serum samples from control sheep and animals with early phase or late phase scrapie. A few potential low molecular weight biomarkers were selected by statistical methods and, after a training analysis, a protein signature pattern, which discriminates between early phase scrapie samples and control sera was identified.

Results: The combination of early phase biomarkers showed a sensitivity of 87% and specificity of 90% for all studied sheep in the early stage of the disease. One of these potential biomarkers was identified and validated in a SELDI-TOF MS kinetic study of sera from Syrian hamsters infected by scrapie, by western blot analysis and ELISA quantitation.

Conclusions: Differential protein expression profiling allows establishing a TSE diagnostic in scrapie sheep, in the early phase of the disease. Some proteic differences observed in scrapie sheep exist in infected hamsters. Further studies are being performed to identify all the discriminant biomarkers of interest and to test our potential markers in a new cohort of animals.

Figure 1

Reproducibility of SELDI-TOF MS analysis using a human serum control. A human serum control was fractionated three times on consecutive days and then the organic fraction F6 spotted and analyzed by SELDI-TOF MS in the range of 2-20 kDa on the same day.

Figure 2

Representative fractionated serum SELDI-TOF MS protein profiles of a PRP^ARR/ARR sheep. F1 (pH9), F2 (pH7), F3 (pH5), F4 (pH4), F5 (pH3), F6 (organic wash).

Figure 3

Representative SELDI-TOF MS protein profiles of serum samples from three healthy animals and three scrapie sheep. (A) Representation of the 4030 Da protein (S1) contained in F4. (B) Representation of the 4250 Da (S2) protein contained in F6. (C) Representation of the 7475 Da (S3) protein contained in F3. (D) Representation of the 9395 Da (S4) protein contained in F1.

Figure 4

Representative distribution of the intensities of selected biomarkers in the sheep populations. (A) Distribution of biomarkers S1 to S4 in control and EP sheep populations. (B) Distribution of biomarkers S1 and S5 to S7 in control and LP sheep populations. The thick line in the boxes indicates the median value of intensities for each population.

Figure 5

mROC graph representations of sensitivities and specificities of the combination of EP (black curve) or LP (grey curve) biomarkers. The mROC linear equations for decision rule are Z_EP = -1.547 × [S2*] +0.466 × [S3*] + 0.661 × [S4*] + 1.238 × [S1*]; Z_LP = 0.295 × [M1*] + 0.397 × [M5*] + 0.176 × [M6] + 0.399 × [M7*] * with λ_S1= 0.05, λ_S2 = -0.17, λ_S3 = 0.32, λ_S4 = 0.44, λ_S5 = -0.18, λ_S7 = 0.01 where λ is the coefficient of Box-Cox transformation: (S* = (S^λ-1)/λ)

Figure 6

Representative SELDI-TOF MS protein profiles of serum samples from three negative control TSE free sheep and three EP scrapie sheep. (A). Representation of the 4030 Da protein (S1) contained in F4. (B) Representation of the 4250 Da (S2) protein contained in F6. (C) Representation of the 7475 Da (S3) protein contained in F3. (D) Representation of the 9395 Da (S4) protein contained in F3.

Figure 7

Representative fractionated serum SELDI-TOF MS protein profiles of Syrian hamster infected with the 263 K scrapie strain (day 29 post-infection): F1 (pH9), F2 (pH7), F3 (pH5), F4 (pH4), F5 (pH3), F6 (organic wash).

Figure 8

Representative distribution of the intensities of selected biomarkers in Syrian hamsters infected with the 263 K scrapie strain: (A) H1 (12 030 Da), (B) H2 (3895 Da) and (C) H3 (7555 Da) at day 0, day 29, day 57, day 106, day 150 post-infection.

Figure 9

Selection of complementary and selective resins for the 12 030 Da biomarker.

Figure 10

SDS-Page separation and SELDI-TOF MS analysis of the purified 12 030 Da biomarker: 0.5 ml of F6 fraction after overload on 10 μl selective resin was used.

Figure 11

Ovine and hamster transthyretin sequences: peptides identified by LC-MS/MS are highlighted. (+) represent amino acids with similar physico-chemical properties.