Ethics of Research involving animals
The transmissible spongiform encephalopathies
6.11 The TSEs are a cluster of degenerative brain diseases. The prototype TSE is scrapie in sheep, but a range of TSE diseases affect different species, including humans. Kuru is a human TSE that was once endemic in New Guinea. It was transmitted by ritualistic cannibalism, which involved eating the brain tissue of other people. The most common TSE in humans is Creutzfeld–Jakob disease (CJD), which occurs sporadically in the human population, with an annual incidence of about one person per million.15 Kuru was the first human TSE that was shown to be transmissible and this was achieved by injecting brain material from patients into chimpanzees. A similar approach showed that CJD could be caused by a transmissible agent, whereas most other neurodegenerative diseases, such as Alzheimer’s disease or Parkinson’s disease, are not transmissible.
6.12 In 1986, a new TSE disease, BSE, was recognised in cattle. It reached epidemic proportions in the UK in the following few years, leading to over 180,000 cases. The origins of BSE have never been established, but it is thought that the epidemic was caused by the nowprohibited practice of feeding ruminant-derived meat and bone meal (MBM) to ruminants as a protein supplement. Evidence of infection with the BSE agent also appeared in zoo animals that had been fed MBM or bovine carcasses, and in domestic cats, which had presumably consumed bovine products in cat food and developed a feline form of BSE.
6.13 In 1996, the first human cases of a new type of TSE, known as vCJD, were observed in young people in the UK. The causative agent of vCJD was shown to be indistinguishable from the BSE agent and infection was presumed to have been caused by eating BSE-contaminated food. By April 2005, 155 cases of definite or probable vCJD had been confirmed in the UK with an average age of onset of clinical symptoms of 29 years of age, and median duration of illness of 14 months, leading to death.16 As the incubation period of TSEs can last for many years, the extent of human infection with the vCJD agent is unknown. For Kuru, the average incubation period was approximately ten years, but in some cases it exceeded 40 years. Thus, human cases of vCJD may continue to appear well into the 21st century. The BSE epidemic in cattle and the sudden appearance of previously unrecognised TSEs in humans and other species led to an unprecedented focus on experimental animal models of these diseases.
The prion hypothesis
6.14 For many years, the nature of the agent that caused TSEs was unknown. Research showed that they were not caused by classical infectious agents, such as viruses or bacteria. Lack of evidence that any form of DNA or RNA was involved led to the development of the prion hypothesis. According to this theory, TSEs were caused by a replicating abnormal form of a protein (a prion), which imprinted its configuration on normal molecules. This would allow prions to be transmitted between animals or humans, causing the disease. This novel hypothesis has subsequently been supported by a large number of experiments, most of which involved inducing TSE in animals.17
Animal models for TSEs: understanding the disease process
6.15 The pathogenesis of TSE diseases is complex and involves transfer and replication of the infectious agent (a prion), which spreads to the CNS via the blood or nerves. Prions do not induce an immune response. The pathology involves the accumulation of abnormal prion proteins in the brain and lymphoid tissues, and the degeneration of nerve cells (spongiosis). The pathogenesis of these diseases cannot be studied in vitro as they involve various physiological systems such as the alimentary tract, lymphoid tissue, nerve routes, peripheral ganglia and various brain regions.
6.16 One of the major steps in the study of the pathogenesis of TSEs was the development of experimental mouse models for the sheep disease scrapie, which had long been recognised as being transmissible between sheep. Transmission of the scrapie agent to mice (by injection of an extract of infected brain tissue from affected sheep into the brain) led to the development of a series of mouse models for scrapie. They were used to identify significant stages in the development of this disease and in defining the different strains of the infectious agent. These studies established that the agent was an abnormal form (PrPsc) of a normal protein (PrP). GM mice in which the PrP gene had been knocked out (see paragraph 5.19) were found to be completely resistant to scrapie, as there is no PrP protein for the PrPsc protein to convert to prions. With regard to welfare implications, mice involved in research on the developmental stages of scrapie typically experienced progressive neurological dysfunction, behavioural and gait abnormalities as well as weight loss. Researchers aimed to limit suffering by euthanising animals at the stage when they were unable to eat or drink without assistance. In some cases, animals were euthanised when they reached certain stages that were known to precede the experimentally induced terminal disease.18 Other welfare implications may arise from the fact that some mice used in this type of research are allowed to age. They may therefore show signs related to old age, such as abscesses, starey coats (not lying flat) or holding their tails abnormally.
6.17 Similar experimental studies have demonstrated the transmissibility of BSE between cattle, sheep and primates. Transmission of BSE to monkeys by injection of bovine prions into the brains of macaques was the first demonstration that BSE was able to cross the species barrier from ruminants to primates. These experiments, undertaken in 1996 in the UK and France, were a forewarning that BSE might be transmissible to humans.
6.18 As there is no immune response to prion infection, it has not yet been possible to develop diagnostic tests that demonstrate the presence of the disease before symptoms occur. Although there is now a range of biochemical markers for detecting the abnormal protein in potentially affected tissues, infection of mice remains the accepted standard for diagnosing prion diseases.
6.19 In view of the potential number of human cases, it is important to develop intervention strategies aimed at slowing down or preventing the spread of prions. This may eventually be achieved by treatment with medicines or through the development of a vaccine. A vaccine could theoretically stimulate the production of antibodies to PrPsc, thus preventing prion proteins from spreading in vivo. Scientists using animals in research with this aim assert that the development of effective therapeutic strategies is likely to depend on continued research on animals.
The contribution of animal models for TSEs to public health policy
6.20 The in vivo models for the pathogenesis of TSEs have had decisive influence on the development of policies for public health aimed at controlling these diseases in cattle and sheep, and to protect humans from further exposure to agents of animal TSEs.19 The current public health measures are based on evidence obtained from experiments on the pathogenesis of TSEs in cattle, sheep, pigs and chickens. Without these studies, it would have been difficult to know how to devise and implement public health measures, other than to prohibit the eating of any animal products, since, at the time, researchers were not able to undertake the research by alternative, non-animal methods.
BSE pathogenesis and public health measures.
6.21 In several large-scale studies on the pathogenesis of BSE, scientists infected calves by feeding them with an extract of cow brain taken from an animal with the disease. The spread of infectivity was then monitored in various tissues. Infectivity was determined by administering extracts of tissue to mice and assessing if they develop the disease (see paragraph 6.18). Several hundred cattle and several thousand mice were used in these experiments. These studies established unequivocally that BSE replicates early on in the gut lymphoid tissues and then spreads to other lymphoid tissue and via major nerves to the CNS. The highest levels of infectivity were found in gut-associated lymphoid tissue, major nerves in the head and neck, brain, spinal cord and collections of nerve cells embedded in the vertebral column known as the dorsal root ganglia. Little infectivity was detected in skeletal muscles.20
6.22 The results from these and many similar studies on the pathogenesis and transmission of TSE between animals have been used to develop policies for public health to prevent the transmission of BSE from cattle through the human food chain. Specifically, they led to the banning of bovine offal for human consumption, the removal of brain, spinal cord and dorsal root ganglia, and the deboning of beef intended for public consumption. Based on knowledge of the dynamics of the spread of prions in vivo, the pathogenesis studies also provided the evidence for the development of the initial Over Thirty Month Scheme (OTMS), whereby the UK Government was able to purchase, for slaughter and ultimate destruction, cattle which were over 30 months of age. This implemented EU Regulations that ordered the prevention of the sale of beef from cattle over this age for human consumption in the UK. The OTMS was a crucial element of legislation for public health, and it may well have averted a larger number of vCJD cases than experienced so far.21
BSE pathogenesis studies in sheep – a model for vCJD
6.23 Sheep are susceptible to infection with the BSE agent, and the dynamics of infection and spread of prions in peripheral tissues is similar to that of vCJD in humans. Thus sheep are commonly held to be a useful model for vCJD. Studies of scrapie in sheep were the first to show that prions could accumulate in the tonsils, and this was shown subsequently to be the case for vCJD. There followed an analysis of the prevalence of vCJD in the human population through retrospective studies on tonsils, and later appendices. The results provided the first information on the number of people that could be incubating the disease.
6.24 BSE pathogenesis studies in sheep also showed that blood can be infectious. BSE can be transmitted between sheep by blood transfusion and current experiments are aimed at identifying the blood fraction that contains infectivity. Scientists conducting these experiments are also interested in exploring the implications of human-to-human transmission of vCJD through blood and have guided UK policy for public health by limiting the potential for this type of spread of vCJD. In 2003, it was found likely that two people who died of vCJD were infected by blood transfusions. As a result, the Department of Health announced in 2004 that anyone who had received a blood transfusion in the UK since 1980 would no longer be able to donate blood themselves.22
Footnotes12 Vilcek and Feldmann M (2004) Historical review: cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci
25: 201–9.
13 In a further series of experiments involving the mouse collagen arthritis model, it was shown that the severity of chronic
arthritis could be reduced with a combination of anti-TNF antibodies and antibodies against T cells. There later followed a
Phase III clinical trial combining anti-TNF treatment with a conventional immunosuppressive treatment to inactivate T cells
in the joint lesions. As in the case of the studies in mice, this refinement of anti-TNF therapy proved successful in halting the
progressive degenerative changes in the joint cartilage and bone in affected joints in patients who were resistant to
conventional drug-based treatment.
14 Vilcek and Feldmann M (2004) Historical review: cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci
25: 201–9.
15 Three forms of the disease had been recognised prior to 1986, sporadic, inherited and iatrogenic (acquired through medical
intervention). See The BSE Enquiry (2000) Report of the BSE Enquiry, Volume 2, Chapter 2, available at:
http://www.bseinquiry.gov.uk/report/volume2/chaptea2.htm#817773. Accessed on: 12 Apr 2005.
16 The National Creutzfeldt–Jakob Disease Surveillance Unit (2005) CJD Statistics, available at:
http://www.cjd.ed.ac.uk/figures.htm. Accessed on: 12 Apr 2005; World Health Organization (2002) Fact sheet: Variant
Creutzfeldt–Jakob disease, available at: http://www.who.int/mediacentre/factsheets/fs180/en/. Accessed on: 12 Apr 2005.
17 For a description of the identification of BSE as a TSE, see The BSE Enquiry (2000) Report of the BSE Enquiry, Volume 2,
Chapter 2, available at: http://www.bseinquiry.gov.uk/report/volume2/chaptea2.htm#817773. Accessed on: 12 Apr 2005.
18 As defined in, for example, Dickinson AG, Meikle VM and Fraser HJ (1968) Identification of a gene which controls the incubation period of some strains of scrapie agent in mice Comp Pathol 78: 293–9; Thackray AM, Klein MA, Aguzzi A and Bujdoso R (2002) Chronic subclinical prion disease induced by low-dose inoculum J Virol 76: 2510–7.
19 Experimental transmission studies in pigs and chickens, for example, showed that these animals are not susceptible to BSE
when fed infected tissue, thus allaying fears that pigs and poultry, which were also exposed to infective MBM, could be
infectious for humans through the food chain.
20 The BSE Enquiry (2000) Report of the BSE Enquiry, Volume 2, Chapter 3, available at:
http://www.bseinquiry.gov.uk/report/volume2/chaptea8.htm#821257. Accessed on: 26 Apr 2005.
21 In 2004–5, the UK Government is implementing a transition towards replacing the OTMS with testing for BSE in cattle of
over thirty months of age. See Department for Environment, Food and Rural Affairs (2005) BSE: Public health issues – Over
Thirty Month cattle – FSA review of the OTM rule, available at:
http://www.defra.gov.uk/animalh/bse/publichealth/otm/review/index.html. Accessed on: 26 Apr 2005.
22 National Blood Service (2004) Variant CJD and blood donation, available at: http://www.blood.co.uk/pdfdocs/vcjd.pdf.
Accessed on: 26 Apr 2005.
23 Farci P (2002) A commentary on the original Science paper (Choo QL, Kuo G, Weiner AJ et al. (1989) Isolation of a cDNA
clone derived from a blood-borne non-A, non-B viral hepatitis genome Science 244: 359–62) J Hepatol 36: 582–5.
24 Farci P (2002) A commentary on the original Science paper (Choo QL, Kuo G, Weiner AJ et al. (1989) Isolation of a cDNA
clone derived from a blood-borne non-A, non-B viral hepatitis genome Science 244: 359–62) J Hepatol 36: 582–5.