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Determination of abnormal prion protein in the milk of cattle infected with the BSE agent

Project Code: M03016

31/08/2005

Veterinary Laboratories Agency, New Haw
Everest, S ; Thorne, L; Jenkins, R; Hammersley, C; Ramsay, A; Hawkins, S; Venables, L; Flynn, L; Sayers, R;
ADAS Rosemaund
Kilpatrick, J;
ADAS Drayton
Sachs, A;
Veterinary Laboratories Agency, Lasswade
Hope, J;
Veterinary Laboratories Agency, New Haw
Jackman, R

Milk and milk products occupy a significant position in the human food chain, from early childhood to old age. In order to support its work on consumer protection, The Food Standards Agency needs to base its decisions, advice and policies on the best available science and as far as milk is concerned, the risk to the human population from animal transmissible spongiform encephalopathies remains undetermined.

Indirect evidence from the epidemiology of maternal transfer in cattle indicates a low risk, but direct analytical evidence for the presence of the infectious agent (as abnormal prion protein) is not yet fully available. This is particularly so with reference to large milk sample volumes and the possible presence of infectivity during the course of BSE. At the present time, milk is the only food source which continues to be obtained from animals developing BSE or even in terminal disease and, as such, requires an analysis of the risk based on analytical data for effective policy control measures to be considered. This project sought to provide that data by utilising the most sensitive PrP^bse detection procedures available, with the results also providing direct experimental evidence for the involvement of milk in any maternal transfer of the disease and thus for DEFRA policy for the control of BSE itself.

Milk specimens were collected from lactating cows, which had previously been challenged with BSE-infected brain at 4 – 6 months of age. One group of 10 animals received a single oral dose of 100g, a second group 1 g and the third was made up of unexposed controls. The cows were artificially inseminated and calved at approximately 2 years of age and annually thereafter. Milking was done within the first week following calving and at 10-weekly intervals during the lactation period. Specimens were centrifuged to obtain a fraction enriched for somatic cells, microbial flora and milk solids and these fractions were analysed for disease-associated, abnormal prion protein (PrP^bse) using a modified commercial BSE ELISA and a different confirmatory assay. No abnormal prion protein has so far been identified in the cellular fraction of milk from cattle incubating BSE using these methods at theirlimits of detection.

The validation of a milk prion assay in the absence of a positive control, naturally- “infectious” milk specimen poses several problems of experimental design. For example, how can the response level of a rapid screening test at which a sample is termed “reactive” - that is, a putative positive test result - be determined without a positive milk standard? This “reactive” signal might be simply close to the high end of a statistical distribution of negative signals rather than necessarily having any deeper significance in terms of association with infection. This problem might be overcome by analysing thousands of true negative samples to determine defined levels of confidence that a “positive” result was a chance false positive rather than a true positive result. Alternatively, a second test based on an independent physicochemical principle could be designed to confirm “reactives” as true negative or positives. We adopted the latter approach to the problem of “reactive” confirmation.

The specificity of the Bio-Rad Platelia™ for PrP^BSE rather than PrP^C is based on the relative resistance of the abnormal protein to hydrolysis by proteinase K under mild denaturing conditions. Therefore, we developed a confirmatory test that excluded proteinases and instead achieved this specificity by physical adsorption of aggregates of PrP^BSE to a polyanionic ligand, the Seprion reagent (Microsens Ltd), followed by elution and detection by a PrP immunoblot system; PrP^C has a much poorer avidity for this ligand under the assay conditions.

The quantitative assessment of risk of BSE transmission to humans via milk requires that any biochemical limit of detection of the screening and confirmatory tests for abnormal BSE prion protein need to be calibrated against an infectivity titre. In the absence of an “infectious” milk control, the VLA “attack rate” study provides the most appropriate data for calibration and is being accrued from experiments where cattle were fed dilutions of infected cattle brain homogenate (Wells, et al., 1994; 1996; 1998; 1999). Similar (but not the same) homogenates from clinical, confirmed cases of BSE were used to produce dilution curves from which limits of detection in mg equivalents of cattle brain were derived and converted into cattle oral ID₅₀ units.

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