NR ASEY
AU Vandevelde,M.; Doherr,M.
TI Official response of the Swiss Reference Laboratory for Animal TSE to the article "BSEs hidden horror" by Debora MacKenzie (http://newscientist.com; June 13, 1998)
QU Mailingliste BSE-L, 12.6.1998
VT
The Swiss reference laboratory for animal TSE (reference lab) is located at the Institute of Animal Neurology at the University of Bern. In order to determine true incidence of bovine spongiform encephalopathy (BSE) in Switzerland, the reference laboratory has been contracted by the Swiss government to monitor TSE's and to conduct studies on the presence of preclinical BSE in the Swiss cattle population. These studies included the development and validation of BSE tests, development of transgenic mice, sampling and examining CNS material from Swiss cattle and epidemiological investigations. The reference lab is not part of the Swiss Federal Veterinary Office as indicated in the article by Deborah MacKenzie of the New Scientist of Today.
We are dismayed by the serious inaccuracies in the article about the "shocking" BSE test results in Switzerland. The interpretation of the results in respect to the validation of the diagnostic tests and true incidence of BSE in the cattle population in our country is unscientific and potentially misleading. We hereby present a brief account of the presented errors and the scientific data and conclusions derived from our studies to date.
Critical statements in New Scientist article to be addressed
1. "Hundreds of thousands of apparently healthy cattle could be infected with BSE, new Swiss data suggest. For every case of mad cow disease in Switzerland, more than 100 animals may be "silenty" carrying the infection"
2. ""The official theory is that only sick cows ate a lump of infectious feedÄ.but other cattle may be infected, and just havent shown symptoms". The Prionics test has confirmed this. Of 1761 healthy cows slaughtered in the culling programme, eight tested positive for BSE. Six of these were also picked up by the veterinary office using other tests."
3. "Eight infected cows out of 1761 gives a rate of "silent" infection of 4.5 per thousand animals, more than 100 times Switzerlands 1997 rate of clinical BSE."
4. "Epidemiological data suggest that a similar pattern may emerge."
Scientific background and response
In 1997, all herd mates of Swiss BSE cases born before December 1990 (the date of the Swiss ruminant feed ban) and all herd mates of Swiss BAB cases (regardless of age) were culled. These animals were examined in the reference laboratory by histology and immunocytochemistry (IHC) to demonstrate the accumulation of the protease resistant (infectious) prion protein. Immunocytochemistry has been used to detect a variety of antigens, including prion protein, in tissue sections since many years. The latter technique involves antibodies specifically directed against the bovine prion protein which we had raised in rabbits. It is known from experimental pathogenesis studies in BSE in cattle that accumulation of the infectious prion protein becomes detectable by immunocytochemical methods before spongiform change and clinical signs occur. Preliminary data suggest that this may be up to 6 months prior to the occurrence of clinical signs (Wells et al., 1998). Of the animals tested, 6 were found to have accumulation of the infectious prion protein in the brainstem. Three of those in addition had mild spongiform changes.
All samples were made available to the company Prionics (B. Oesch, M. Moser) during a validation phase of a BSE test developed by Prionics at the University of Zuerich. The Prionics test is also based on the detection of infectious prion protein with western blot (WB) techniques using antibodies following electrophoresis of brain tissue extracts. The WB technique exists since many years and is widely used in the biomedical sciences. The use of the WB for the detection of prion protein in BSE was first published in 1992 in Great Britain but remained essentially a research tool. Prionics has modified this technique in such a way that large numbers of samples can be examined in a short period of time, thus allowing its use as a routine diagnostic test. Prionics has also developed excellent monoclonal antibodies against the infectious prion protein.
The WB validation also included confirmed cases of BSE as well as confirmed negatives. The results were evaluated by Prionics together with the reference laboratory and a final report written and signed by the two laboratories in spring of 1998 was submitted to the Swiss FVO. The Prionics test detected 6 positive cases among the 1761 animals culled in the slaughter programme. Four of these cases were identical to the ones found by the reference laboratory. The 2 remaining cases detected by Prionics had been negative in the IHC. In contrast, the 2 additional cases that were detected with standard methods were negative in the Prionics assay. Thus a total of 8 positive reactors were found, 4 of which overlapped. Both Prionics and the reference laboratory agreed in the final report to declare the 4 overlapping reactors definite positive cases. The remaining 4 animals were considered questionable, requiring further investigation. Meanwhile, all 8 reactors have been examined, again in a blinded fashion, in the laboratory of Prof. Kretschmar, University of Güttingen, Germany using a new very sensitive assay in tissue sections. The new assay confirmed the diagnosis in one of the two questionable cases diagnosed by the reference laboratory. The two questionable cases diagnosed by Prionics were not confirmed. In total, 5 out of 1761 herdmates of confirmed BSE cases were confirmed PrP positive and considered preclinically infected.
It is of no surprise to scientists that for any disease with a long incubation period (time between infection and outbreak of clinical signs), one has to expect a larger proportion of the infected population to be in the incubation period (preclinically infected) than showing clinical signs. This is especially true in the case of BSE where the exposure to contaminated meat and bone meal (MBM) occurred over a period of several years and where the incubation period can vary considerably between individual animals: Swiss BSE field cases were between 31 months and 11 years old with an average of 5-6 years.
Until recently, no studies have been reported on the prevalence of preclinical BSE in the slaughter animal population. The limiting factor for these studies remains the fact that the tests currently available only detect animal in the late incubation period (within 6 months of the outbreak of clinical disease). Estimates of preclinically infected animals that were slaughtered on a routine basis and that entered the food chain undetected are based on modelling approaches. A mathematical model developed in 1996 in Great Britain on the basis of the MAFF BSE statistic estimated that a large number of preclinically infected cattle had already entered the human food chain in the UK (Anderson et al., 1996).
A backward calculation model on the expected number of animals that had to be infected during their first year of life to produce the number of observed cases from each birth cohort since 1984 (the birth year of the first BSE case observed in Switzerland) was performed for the Swiss BSE epidemic. We estimated that the maximum number of preclinically infected animals in Switzerland was reached in 1990 (with potentially 1200 affected animals among the 900000 heifers and cows in the population that year). After 1990, most likely as a result of the feedban, the model shows a pronounced downward trend in the expected number of preclinical cases in the general cattle population with less than 100 preclinical cases still present in the 1997 population. Each year, approx. 70000 heifers and 200000 cows are slaughtered in Switzerland.
The results of these studies should be interpreted as follows:
- The sensitivity of the Prionics WB is similar to the one of the IHC and certainly not higher as suggested by the media. With the sensitivity of the techniques available to date, animals in incubation (preclinical cases) will be detected by demonstration of the accumulation of the protease resistant protein in the final 6 months of the incubation period only;
- Demonstration of accumulation or infectious prion protein, even in the absence of clinical signs and spongiform changes, is a strong indication for BSE; however, as with every test system, false positive results are possible;
- In contrast to the statement made by D. MacKenzie, researchers have always suspected a large number of preclinically infected animals in the population. For that reason, measures have been taken (destruction of slaughter animals over the age of 30 months in the UK, limitation on the use of high risk organs like brain and spinal cord) to prevent the spread of infection from products of slaughtered preclinically infected animals to humans;
- Of the 1761 herd mates of BSE cases destroyed in the culling programme, 5 positive in IHC and the new assay, i.e. in the late stage of incubation, not 8 as stated in the article. It is incorrect that these preclinical cases of BSE were detected (the first time) by the new "sensitive" assay of Prionics; the cases had been identified before in the frame of a study conducted by the reference lab and, together with other samples, were used for the validation of the Prionics western blot.
- We consider the culled population of BSE herdmates a high risk group with proven exposure. The prevalence of preclinical disease observed within this cohort should therefore be considerably higher than the true prevalence of preclinical BSE cases in the general cattle population. Any direct extrapolation showing huge numbers of preclinically infected animals for 1997, as done by D. MacKenzie, is not appropriate;
- As demonstrated before in the Swiss simulation, we expect (with advancing time) a further decreasing proportion of preclinically infected animals in the Swiss cattle population. This is due to elimination of exposure (feed ban and similar measures) and the decrease in animals from exposed birth cohorts that are still present in the population (aging of the cohorts);
- We currently have no epidemiological data to support that "a similar pattern" (high prevalence of preclinical infection) may emerge in the planned prevalence study at slaughter, as incorrectly stated by D. MacKenzie.
Supporting our line of reasoning is a statement published by the moderator of ProMED-mail (e-mail: promed@usa.healthnet.org) as a response to D. MacKenzies article:
"One should remember that these 1761 healthy cows were taken from herds in which one or more clinical BSE cases had been previously identified and slaughtered. And 6/8 Prionic +ve animals showed positive on other veterinary tests suggesting that if they had not been slaughtered some if not all six would have eventually demonstrated BSE. It has been held in the UK for some time now that preclinically affected animals would have been slaughtered and butchered as a matter of course. What risk such animals pose is argumentative with the current restrictions. - Mod.MHJ"
Prof. Marc Vandevelde
Institute for Animal Neurology, University of Bern, CH-3012 Bern
and Dr. M. Doherr
Institute of Virology and Immunoprophylaxis (IVI), Ch-3012 Mittelhaeusern
References
- Anderson R.M. et al. (1996). Transmission dynamics and epidemiology of BSE in british cattle. Nature 382(N6594): 779-88.
- Wells G.A. et al. (1998). Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Veterinary Record 142(5): 103-106.
IN 1997 wurden in der Schweiz alle Rinder aus Herden getötet, in denen mindestens ein BSE-Fall aufgetreten war. Auch die scheinbar gesunden Tiere wurden vom als Referenzlabor für tierische übertragbare spongiforme Encephalopathien in der Schweiz zuständigen Referenzlabor im Institut für Neurologie der Tiere an der Universität Bern histologisch und immunzytochemisch in Gewebeschnitten mit Kaninchen-anti-Rinderprionprotein-Antikörpern auf Prionproteinakkumulationen hin untersucht. Dabei fielen 6 Rinder mit Prionproteinakkumulationen im Stammhirn auf, von denen 3 auch milde spongiforme Degenerationen zeigten. 1761 Proben der scheinbar gesunden Rinder wurden der Firma Prionics für die Validierung ihres Immunoblot-BSE-Testes zur Verfügung gestellt. Auch der Prionics-Test markierte 6 Proben als BSE-positiv, wobei jedoch nur 4 Tiere vom Referenzlabor und Prionics als positiv erkannt wurden. Vier Proben waren jeweils nur in einem Test positiv. Deshalb wurden die 8 zumindest einmal positiven Proben zusätzlich einem neuen Test der Arbeitsgruppe von Prof. Kretzschmar in Göttingen unterworfen, der sehr sensitiv sein soll und wie der Test des Referenzlabors der Schweiz mit Gewebeschnitten arbeitet. Von den 3 umstrittenen Proben wurde nur eines der beiden im Referenzlabor positiven Ergebnisse bestätigt.
AD Dr. Marcus G. Doherr, Ph.D., c/o Epidemiology Unit, Institute of Virology and Immunoprophylaxis, CH-3147 Mittelhaeusern / Switzerland, Phone: +41 (31) 848 9262 (work) & +41 (31) 972 2661 (home), FAX: +41 (31) 848 9222 EMAIL: Marcus.Doherr@ivi.admin.ch
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SP englisch