title / Extended provision of bovine body fluids from “pre-clinical” BSE and control animals.
/ DEFRA
project code / SE1749
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Extended provision of bovine body fluids from “pre-clinical” BSE and control animals.
DEFRA project code / SE1749
Contractor organisation and location / Veterinary Laboratories Agency,
New Haw, Addlestone,Surrey
Total DEFRA project costs / £ 1,162,244
Project start date / 01/10/97 / Project end date / 31/03/03
Executive summary (maximum 2 sides A4)
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The necessity for a major improvement in TSE diagnostics became paramount following the1996 vCJD outbreak, with the priority for DEFRA policy being the development of live animal tests and tests capable of indicating pre-clinical disease. At this time, few body fluid samples were being archived from terminal BSE cases and there was no opportunity for identifying and collecting from animals incubating the disease but not yet in the clinical phase. This project was hence set up to supply blood, urine and cerebrospinal fluid (CSF) at regular intervals during the disease so that emerging diagnostics could be evaluated and validated.
An initial project SE1737 was initiated in January 1997. Four, 3 months old calves were each challenged orally on 14.01.97 with 100g of a BSE-infected brain pool in a single dose, a quantity sufficient to cause eventual disease. Body fluids were collected and archived throughout the course of the disease, although there was a short delay in the collection of CSF samples due to some welfare difficulties – these were rapidly resolved.
It quickly became apparent that there was a necessity for the production of age matched controls, with project SE1749 being funded , in order to meet this requirement. A further 4 calves were challenged with 100g BSE-affected brain, 4 calves challenged with 100g BSE-negative brain (collected from New Zealand cattle) and 8 age-matched unchallenged controls. The calves were purchased from BSE-free farms that had previously been used to supply animals for transmission and attack rate studies during September and October 1997. These animals were challenged on 27.11.97 with the two projects being combined as of 01.04.01.
During the early stages of this project, 2 possible tests directed towards the live animal were under development at VLA, both of which necessitated body fluids such as those targetted under SE1749. The first test was an electrochemical fingerprinting of urinary metabolites which ultimately, despite initially displaying promising results, failed to generate the required test specificity and hence did not require evaluation against pre-clinical samples.
The second test involved the detection of S-100b protein in CSF. This was a collaborative exercise undertaken between TMB2 and the test’s originator, Dr Alison Green (Institute of Neurology). Initial results had demonstrated differences between clinically affected BSE animals and both clinically normal and BSE-negative clinical suspect controls. S-100b levels determined using the 4 challenged animals from SE1737, during the first 6 months post challenge, increased to levels equivalent to those reached in confirmed BSE cases. The significance of this finding could not be assessed under SE1737 due to the absence of any control matched fluids and, hence the requirement for SE1749. The application of the appropriate controls, however, only served to demonstrate that the rises seen were in fact transient and cyclical, occurring in both challenged and unchallenged animals. Subsequent to this, levels of 14-3-3 Protein in CSF, a useful diagnostic in CJD but of unproven value in BSE, have been determined also by Dr Green who has continued her collaboration following a move to the CJD Surveillance Unit, Edinburgh. The analysis of 14-3-3 protein has also been studied, albeit using an alternative technology, by Prof. H Schroeder at the University of Mainz. Neither analysis has shown direct evidence of diagnostic value in bovine CSF but there is sufficient evidence that the Schroeder technique requires evaluation on a larger set of samples. These have been sourced and supplied.
Three additional analyses have been applied, at the VLA, to the CSF collected under SE1737 and SE1749. These tests were not for use as possible diagnostics, but as baseline determinants to monitor storage and CSF quality. These include total immunoglobulin G (IgG) and protein levels. Both have remained consistent throughout the time course of the study to date, except for occasional significant increases, strongly indicating innapproriate sample collection and / or contamination of the sample with blood. These samples have hence been deemed unsuitable for any future use in TSE diagnostics. It had also been the intention to monitor the brain isoform of creatine kinase both as a potential differential diagnostic and for evidence of trauma in the sampling of CSF. However, levels proved to be too low for use either as a potential diagnostic marker or as a baseline monitor for storage effects. It has always been the intention to complete the biochemical analyses of the samples collected, on termination of the project, that is, when the last BSE challenged animal developed clinical disease. Despite the project being extended by a year, this situation has yet to be reached.
In addition to the analytical assessments carried out, the animals have been clinically observed throughout the experiment. Initially this involved a combination of observations by staff from the Animal Services Unit at VLA during daily husbandry and ad hoc, studies by clinicians from the Neuropathology Unit. From July 1999 onwards, detailed weekly observations and assessments were instigated for the final period of the disease and detailed clinical reports presented. During the course of project SE1737, only 2 of the 4 animals developed BSE. The remaining animals were expected to develop clinical BSE shortly after the conclusion of this project (March 2001) and hence were supported using funding ascribed to SE1749. At the conclusion of the one year extension to SE1749, 3 challenged animals, 4 New Zealand BSE-negative challenged animals and 8 unchallenged animals remain. In the absence of further funding, the cost of maintaining these animals will be attributed to the TSE Archive until these animals are killed. Although no precise date can be predicted for such killing it should be stated that all the challenged animals are showing clinical signs, although there is as yet no progression. The four New Zealand control animals are to be retained until the death of those that have been challenged, with the remaining unchallenged controls being kept until the end of May 2003 at which time they will be culled.
As of the time of writing this report, the BSE-negative challenged animals remain healthy and show no signs of BSE (CM880, CM879, CM866, CM861). None of the environmental controls (CM862, CM863, CM868, CM870, CM871, CM874, CM875) was killed in 2002/2003. Of the three remaining dosed steers (CM864, CM865, CM873), all display possible signs of BSE but with no further progression to date. CM869 was killed with signs of BSE on 14/05/02 and was subsequently confirmed by histopathology/ICC. The clinical report can be found attached (annex 1).
During the preparation of this report, CM873 developed final clinical phase symptoms and was necropsied in extremis on 19.09.2003. No confirmatory diagnosis is yet available but a completed case history will be communicated to defra as soon as it becomes available.
It has been decided that this project will not be extended beyond 31.03.03 and that the archiving and analysis of the remaining samples will be undertaken using TS5700, TSE Archiving, funding. The contents of this final report are inevitably similar to the final report prepared at the conclusion of the last financial year (2001-2002), with the only differences being the status of the animals, the latest total protein and IgG results and the storage of the last year’s samples.
CSG 15 (1/00) 2
Projecttitle / Extended provision of bovine body fluids from “pre-clinical” BSE and control animals.
/ DEFRA
project code / SE1749
Scientific report (maximum 20 sides A4)
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CSG 15 (1/00) 2
Projecttitle / Extended provision of bovine body fluids from “pre-clinical” BSE and control animals.
/ DEFRA
project code / SE1749
All calves for SE1749 were sourced from BSE non-exposed herds, brought into VLA-Weybridge during September and October 1997 and challenged on 27.11.97, following health checks and a period of acclimatisation. Subsequent mouse bioassay of the BSE-positive inoculum gave a titre of 102.86 log10 mouse i.c/i.p LD50/g of tissue.
Throughout the project, weekly monitoring has been in progress. Intercurrent minor ailments and necessary treatments have been noted and logged. All animal movements necessitated by accommodation requirements have also been logged. SE1737 animals which were incorporated into SE1749 at 01.04.01 were challenged on 14.01.97.
Samples of body fluids (primarily blood, CSF and urine) have been collected at regular intervals throughout. Serum obtained from clotted whole blood has been harvested and archived as has buffy coat / leucocyte samples prepared from heparinised whole blood and subsequently separated by centrifugation in Ficol / Histopaque. Initially, CSF was collected and frozen without prior treatment but contacts with groups such as Prof Schroeder (University of Mainz), prompted a change in sample preparation in line with human clinical biochemistry practice. As from 16/12/1999, half the CSF collected has been archived untreated, and half centrifuged to remove cellular material prior to freezing.
All samples have been logged on receipt providing details of date received, date taken, animal number, project code, condition on arrival, receiving officer, sample type and any general comments. Prior to April 1998, the log book and archive (Low Temperature Storage Facility – LTSF) details were the source of information. From 22.04.1998 an additional record including amount of sample taken, volume of sedative (Rompun) used and more detailed notes of CSF collection has been produced and a unique in-house sample numbering system incorporated. Up to and including March 2002 the extensive library of samples taken, their logging-in, preparation and storage details have been reported in previous CSG12 Annual Reports for SE1749 and the CSG15 Final Report for SE1737. Annex 3 contains all the information on the samples collected during 2002 / 2003. This is a large amount of information in toto and on termination of the project or when the last animal goes down with BSE a CD of sampling details will be prepared for annexing to the final report.
The biochemical analyses carried out to date have been confined to CSF. S-100b Protein has been the major project target for a potential diagnostic. Analyses have been carried out by Dr Alison Green, originally at the Institute of Neurology, London and more recently at the CJD Surveillance Unit in Edinburgh. Previous studies (Green & Jackman 1999 Vet Rec 145, 107 – 109) had demonstrated a significant ( P<0.006) rise in S-100b levels in animals with confirmed BSE when compared to clinical suspects without BSE. CSF S-100b Protein levels in animals CL645, CL646, CL648 & CL651 were determined on an ongoing basis and showed an unexpected rise around 24 – 28 weeks post challenge and later at 35 – 40 weeks. At this time it was not possible to ascertain the diagnostic significance of this finding as there were no appropriate controls allowed within that project. Within SE1749, similar transient rises have been observed irrespective of BSE status during the early developmental stages of the animals. As the animals have progressed towards clinical disease, no significant rises in S-100b levels have been observed – contrary to the data seen in BSE field cases. Data for the changes in levels of S-100b Protein in the CSF of those animals followed through to termination are given in Annex 2, figs 1, 2 and 3 attached.
A second possible surrogate marker, 14-3-3 Protein has been followed on a smaller scale during the study involving the methods at the National CJD Surveillance Unit, Edinburgh (Dr Alison Green) and at the University of Mainz (Profs Schroeder and Muller). Annex 2, fig 4 demonstrates that the application of the ELISA at the NCJD Unit provides little of diagnostic value in BSE in contrast with its use in CJD diagnosis.
Similarly, the evidence provided by Prof Schroeder - presented in its entirety at Annex 2 Figs 5a – 5m
-is not clear cut, but it does indicate that this warrants an extended evaluation on further samples from this study.
Other parameters followed during the study were total protein and immunoglobulin G (IgG). These were selected as markers of sample quality, not expected to change as a result of disease and which would be used to highlight problems with contamination of samples during collection and diagnosis of infection within the CSF of the animal. They would also assist in confirming that storage of samples were not compromised. IgG has been determined by a direct ELISA with an analytical sensitivity (defined as the minimum concentration giving an optical density greater than three times the standard deviation of the blank) of <0.002ug ml-1. The standard curve is linear to 0.125ug ml-1 (r2=0.996). Graphic representations of the IgG profiles to date are shown in Annex 2 Fig 6a.
Total protein content of the CSF has been assayed using the Pierce BCA (bichiconinic acid) kit which has an analytical sensitivity of <3.1ug ml-1 and a linear standard curve to 100ug ml-1 (r2=0.994). Data for the 3
groups of cattle is presented in Annex 2, Fig 6b.
All data has been plotted against the same timepoints post challenge. Given the variable incubation
periods expected it may be more appropriate to present the values against a pre-determined percentage of
disease duration. In addition, the Total Protein and / or Total IgG levels may be used as determinants for
normalising the S-100b data.