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. 2005 Jul;12(7):814-20.
doi: 10.1128/CDLI.12.7.814-820.2005.

Bov-tA Short Interspersed Nucleotide Element Sequences in Circulating Nucleic Acids From Sera of Cattle With Bovine Spongiform Encephalopathy (BSE) and Sera of Cattle Exposed to BSE

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Bov-tA Short Interspersed Nucleotide Element Sequences in Circulating Nucleic Acids From Sera of Cattle With Bovine Spongiform Encephalopathy (BSE) and Sera of Cattle Exposed to BSE

Ekkehard Schütz et al. Clin Diagn Lab Immunol. .
Free PMC article

Abstract

Circulating nucleic acids (CNA) are known to be enriched in repetitive DNA sequences in humans. Here, bovine sera CNA were analyzed to determine if cell stress-related short interspersed nucleotide elements (SINEs) could be detected in sera from cattle associated with bovine spongiform encephalopathy (BSE). Nucleic acids were extracted, amplified, cloned, and sequenced from the sera of protease-resistant prion protein (PrP(res))-positive cattle (n = 2) and sera from BSE-cohort cows (n = 6); 150 out of 163 clones revealed the presence of, on average, an 80-bp sequence from the 3' region of Bov-tA SINE. A PCR protocol was developed that differentially identified SINE-associated CNA in BSE-exposed versus normal cattle. CNA were extracted from a serum vesicular fraction after controlled blood collection and processing procedures. Sera from four confirmed cases of BSE, 137 BSE-exposed cohort animals associated with eight confirmed BSE cases, and 845 healthy, PrP(res)-negative control cows were tested. All four sera from confirmed BSE cases were repeatedly reactive in the assay. BSE-exposed cohorts had a 100-fold higher occurrence of repeatedly reactive individuals per cohort (average = 63%; range = 33% to 91%), compared to healthy controls (average = 0.6%; P < 0.001). This study shows that BSE-confirmed and cohort animals possess a unique profile of SINE-associated serum CNA that can be utilized as a marker that highly correlates to BSE exposure.

Figures

FIG. 1.
FIG. 1.
Upper panel. Post-PCR (CHX-1F/CHX-1R) PAGE analysis of sera from two BSE cattle, four BSE-exposed cohort animals, and three normal controls. Lanes 1 to 3: normal control samples N1 to N3; lanes 4 to 7: cohort samples C1 to C4; lanes 8 and 9: PrPres-positive BSE cases BSE1 and BSE2; lane 10: size markers with sizes shown at the right. Lower panel. Melting curves from the same experiment as shown in the upper panel; 30 cycles of PCR with primers CHX-1F and CHX-1R were performed. The differences between cohort (C1 to C4) and BSE (BSE1 and BSE2) samples within the diagnostic range (87 to 90°C) are statistically significant (P < 0.01) versus the nontemplate control (NTC) and versus normals (N1 to N3). The AUC values are shown in parentheses.
FIG. 2.
FIG. 2.
DNA sequence alignments (5′ to 3′, left to right) from three individual CNA fragments derived from PCR with the CHX-1F and CHX-1R primers. A common element identified from two confirmed BSE cases (CNA4), depicted as a solid gray box, in all CNA fragments is homologous to the monomer region of the Bov-tA SINE sequence (accession number X64124). The 5′ Bov-tA-like sequence in CNA4 is followed 3′ downstream by homologous fragments derived from accession number AC092496. CNA6 is a sequence identified from BSE cow 1 and cohort sera. The 5′ Bov-tA-like sequence is followed 3′ downstream by homologous fragments derived from accession number AC091728.2. CNA1 is a sequence identified from BSE cow 2 and cohort sera. The 5′ Bov-tA-like sequence is followed 3′ downstream with homologous fragments derived from accession number AC091660.2. Open boxes are plus/plus homologies (11 to 20 bp); diagonally striped boxes are plus/minus homologies (11 to 20 bp).
FIG. 3.
FIG. 3.
Reactivity of four confirmed BSE cases (solid black bar), eight unrelated BSE cohorts, and healthy control animals. All eight cohorts (diagonally striped bars; cohort numbers are given according to Table 3) showed a higher proportion of repeatedly reactive samples than random healthy controls (solid gray bar), ranging from 33% to 91% of each cohort. Only one healthy control out of 176 was found to be repeatedly reactive (0.6%). The differences between cohorts and an apparently healthy control herd as well as randomly selected cattle without detectable PrPres post mortem are highly significant (P < 0.001).

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