Evaluation of rapid post-mortem test kits for bovine spongiform encephalopathy (BSE) screening in Japan: Their analytical sensitivity to atypical BSE prions

Abstract

A classical type of bovine spongiform encephalopathy (C-BSE), recognized in 1987, had a large impact on public health due to its zoonotic link to variant Creutzfeldt-Jakob disease by the human consumption of dietary products contaminated with the C-BSE prion. Thus, a number of countries implemented BSE surveillance using rapid post-mortem test kits that were approved for detection of the C-BSE prion in the cattle brain. However, as atypical BSE (L- and H-BSE) cases emerged in subsequent years, the efficacy of the kits for the detection of atypical BSE prions became a matter of concern. In response to this, laboratories in the European Union and Canada evaluated the kits used in their countries. Here, we carried out an evaluation study of NippiBL®, a kit currently used for BSE screening in Japan. By applying the kit to cattle brains of field cases of C-BSE and L-BSE, and an experimental case of H-BSE, we showed its comparable sensitivities to C, L-, and H-BSE prions, and satisfactory performance required by the European Food Safety Authority. In addition to NippiBL®, two kits (TeSeE® and FRELISA®) formerly used in Japan were effective for detection of the L-BSE prion, although the two kits were unable to be tested for the H-BSE prion due to the discontinuation of domestic sales during this study. These results indicate that BSE screening in Japan is as effective as those in other countries, and it is unlikely that cases of atypical BSE have been overlooked.

Keywords: BSE surveillance; ELISA; analytical sensitivity; atypical bovine spongiform encephalopathy; evaluation study; prion; rapid post-mortem test.

Figure 1.

Dilution-response profiles of the kits using the brain homogenate of a C-BSE cow. Raw data were plotted as the mean ± SEM (standard error of the mean) from a set of triplicate wells. The dotted lines indicate thresholds of positivity defined by the manufacturers' protocols. Non-linear curve fitting was applied to the row data using a four-parameter logistic model. (A) TeSeE®, (B) FRELISA®, (C) NippiBL®, and (D) BetaPrion®.

Figure 2.

Dilution-response profiles of the kits using the brain homogenates of L- and H-BSE cows. Raw data were plotted as the mean ± SEM from a set of triplicate wells. The dotted lines indicate thresholds of positivity defined by the manufacturers' protocols. Non-linear curve fitting was applied to the row data using a four-parameter logistic model. (A – D) TeSeE®, FRELISA®, NippiBL®, and BetaPrion® tested on the samples prepared from the L-BSE cow, respectively. (E) NippiBL® tested on the samples prepared from the H-BSE cow.

Figure 3.

Western blot analysis after PK digestion to determine the relative amounts of PrP^Sc in the stock homogenates of the brain. (A) The stock homogenates of the brains of the C-, L-, and H-BSE cows were digested by PK, and aliquots of volumes of the digests corresponding to the indicated weights of tissues were subjected to Western blot analysis. PrP^Sc was detected using anti-PrP antibody 12F10, with the aid of a chemluminescent detection reagent and a cooled CCD camera imaging system. The letters non-, mono-, and di- denote the non-, N-mono-, and N-di-glycosylated forms of PrP^Sc. (B) Signal intensities of the non-, mono-, and di-glycosylated forms of PrP^Sc in each lane in (A) were measured by ImageGuage software, combined as a total signal intensity of PrP^Sc, and plotted in relative magnitude by taking that of 50 μg of the C-BSE brain tissue as 10.0.

Figure 4.

Effect of the sample digestion condition of NippiBL® on detection of the L-BSE prion. (A) Brain samples of the C-BSE and L-BSE cows were digested according to the manufacturer's protocol but for an extended time. For the L-BSE brain, samples of three different dilutions by normal brains were tested (a: 2^−0.6 dilution, b: 2⁻¹ dilution, c: 2⁻² dilution). The arrowhead at the top of (A) indicates the digestion time set by the protocol (10 min). Data were plotted as the mean ± SEM from duplicate wells. The dotted lines indicate the thresholds of positivity defined by the protocol. The samples of normal brain (i.e., negative control) gave rise to negative signals throughout the assay. (B) Only data on the brain samples of the L-BSE cow in (A) were plotted for clarity.