Genetic aspects of Mad Cow Disease (BSE), Scrapie and CJD

Dorian J. Garrick
Institute of Veterinary, Animal and Biomedical Sciences,
Massey University, Palmerston North, New Zealand

Spongiform diseases

There are many mammalian diseases known as Spongiform encephalopathies - so-named because they cause the brain to become riddled with holes (sponge-like). On a global basis, the most common form of these diseases is Scrapie, found in sheep and goats. Afflicted animals lose co-ordination and develop an itch, leading them to scrape off their wool/hair.

Scrapie was thought to have been found in New Zealand from a 1954 importation of Suffolk sheep (subsequently slaughtered from 1956 to 1958) and from 1973 imports of East Friesian sheep that were diagnosed and slaughtered in 1976. It is not known to exist in New Zealand at the present time. In contrast, many European sheep flocks have had a high incidence of this condition for at least 250 years. In some cases, up to one-tenth of the flock can succumb to the disease in any one year.

A number of similar human conditions exist, including Kuru (seen only among some New Guinean Highland tribes known at the time for eating human brains), Creutzfeldt-Jakob disease (CJD) occurring worldwide and evident as mental deterioration, Gerstmann-Straussler-Scheinker (GSS) disease resulting in loss of muscular co-ordination, and fatal familial insomnia, in which dementia follows difficulty sleeping.

The cattle form of the disease, known as Bovine Spongiform Encephalopathy (BSE) or mad cow disease was formally identified in 1986 in Britain and has been since discovered in a number of European countries. Hearsay evidence suggests the disease has been at high levels since the early 1980's if not the late 1970's. The U.K. currently has some 160,000 BSE cases, with over half the dairy herds and one-sixth of beef breeding herds affected. Similar forms of disease are found in cats, mink, deer and other species.

Prions - the causal agents

Most diseases are caused by viral, bacterial or fungal infection. The infectious organisms reproduce to make more copies of themselves, based on genetic material (such as DNA or RNA) contained within the causal organism. Genetic material can usually be degraded by ultraviolet or ionizing radiation as may be used for sterilizing surgical instruments. However, it was discovered that brains from scrapie-infected sheep treated in this way retained their ability to cause the disease when introduced to healthy sheep. Workers in the late 1970's showed that treatment of infected tissue, using procedures that unfolded or degraded protein, reduced infectivity. The disease seemed to be caused by an infectious protein, now known as a prion.

It is not known how prions damage brain cells. However, in tissue cultures the prions accumulate within cells in structures known as lysosomes. It is thought these might burst, damaging the cell. When a diseased cell dies, a hole is created in the brain and the prions are released to accumulate in as yet undamaged cells.

Proteins are manufactured by joining together of amino acids, with DNA storing instructions as to the sequence of amino acids involved. The sequence of amino acids that forms the prion is naturally present in all individuals, with the resulting protein usually clinging to the outside of nerve cells in the brain, without causing development of the disease. This particular protein consists of a sequence of about 250 amino acids which normally twist into a specific kind of spiral, known as an alpha helix. The scrapie-causing form involves the shape of this protein changing to beta sheets, in which the backbone of the protein is fully extended.

Two ways to catch the disease

These diseases may appear sporadically, some appear inherited and others have been spread by medical intervention (particularly by corneal transplant or injection of growth or reproductive hormones derived from pituitaries of cadavers). A test-tube of alpha-helix shaped protein when mixed with the same protein in the beta sheet configuration resulted in the conversion of the alpha-helices to beta sheets. Thus the normal or safe form of the protein can be converted into the prion or infectious form by simply coming into contact with a prion form. The disease will therefore be caused by contracting one or more copies of the protein in the prion or dangerous form. It is then a matter of time (perhaps many years) for the development of the disease as the safe form of the protein is gradually converted into the prion form.

Studies of humans with GSS (which is usually inherited) show that afflicted individuals have a different DNA sequence (resulting from a base pair substitution) which changes one of the amino acids in the protein sequence from leucine to proline. It is believed that this change leads to a less stable form of the protein, such that it can spontaneously change to the prion shape at some stage in the individual's lifetime. Once initiated, this will result in a cascade whereby the normal protein changes to the prion form. The disease therefore seems to be inherited in that some forms of the protein change more readily than others.

In cases where the disease appears to be have been caught, rather than inherited, it is believed to be due to contracting the prion form within the body. The most effective way to pass on the disease is to inject the prion form directly into the brain. Injection into the blood supply would be less effective and eating the prion would be less effective again. The consumption of protein would normally result in the protein being broken into pieces in the stomach or small intestine, with the small pieces (which would be harmless) taken up into the blood stream. In some cases, feeding is believed to have led to the protein being absorbed in the prion form. Perhaps the presence of stomach ulcers, for instance, would allow such uptake to occur. In many cases, heating (such as in cooking) will denature (destroy) proteins. However, it appears that the prion form of the protein is less susceptible to unfolding or denaturing than is the safe form.

Studies of the development of the disease in natural outbreaks compared to sheep innoculated with the prion by alternative routes suggest that in natural outbreaks the disease is maternally transmitted from ewe to lamb via an oral route, although other transmission mechanisms cannot be ruled out.

Confirmation with laboratory mice

Genetic engineering has allowed a mouse strain to be developed in which the gene responsible for manufacturing the protein that can form the prion has been knocked out. These mice do not naturally have the safe or any other form of the protein and are unaffected by introduction of the prion form. This proves that it is the conversion of the individual's own protein into the dangerous form that causes the symptoms of the disease to develop. The function of this protein in its normal form is under investigation but it appears necessary to maintain certain classes of brain cells (Purkinje cells) that are essential for balance and muscular function. The knock-out mice develop wobbly legs and cannot walk in a straight line. In other lab mice where the protein gene has been modified, rather than knocked out, the sleep patterns of the mice are disrupted. Disrupted sleep is an early symptom of fatal familial disease, an extremely rare inherited prion disease.

Selection to reduce the occurrence of these diseases

At least three British flocks have been developed whereby lines of sheep have been selected for or against their susceptibility to scrapie following deliberate innoculation. In one study, infectious material innoculated into the brain of susceptible sheep required 200 days incubation whereas the resistant lines took 900 days incubation to show development of the disease. Prions that were injected under the skin required 300 days incubation in the susceptible line and did not cause infection in the resistant line. These studies have shown that progress can be achieved in lengthening the period from introduction of the prion to full-blown development of the condition. The mechanism of this effect appears to be simply inherited and may reflect selection for a variant form of the normal protein.

Human cases

The incidence of CJD in Britain is slightly higher in dairy farmers and abbatoir workers which may argue that non-oral routes are the basis for transmission - perhaps through the nose or damaged skin. However, unexplained is the fact that the highest incidence of CJD in the U.K. is among vicars. The supposed role of consuming infected beef as the source of infection does little to explain the fact that vegetarians also succumb to the disease. The number of CJD cases in the U.K. has declined from 1994 to 1995, but the incidence in the 1990's has been somewhat higher than in the 1980's. The ten new cases of CJD that led to the recent blockade on beef were unusual in that they occurred in people under 42 years of age. It has been argued that similar cases may have occurred in the past but were mis-diagnosed as meningitis or measles.

Strains of the disease

Experiments have shown that the infectivity of infected brain tissue can be modified by treatment of the tissue. This might suggest that there are more than two shapes that the protein can take on, with the dangerous shapes varying in their ability to encourage normal protein to change shape.

It is well known that it is hard to infect one species (say mice) with prions from another species (say sheep). A research team generated transgenic mice expressing the hamster form of the safe protein. Normal mice inoculated with hamster prions rarely acquire scrapie, but the transgenic mice became ill within two months. It has since been concluded that the more the amino acid sequence of the prion or dangerous form resembles the safe form, the greater the chance of encouraging a shape change. Sheep and bovine safe proteins differ at seven positions and attempts to deliberately infect cattle with scrapie material have been unsuccessful. However, having occurred in one instance, the infected cow would then carry the cattle prion and would be able to infect other cattle more readily. The human and bovine form of the protein differ at more than 30 positions. It has since been shown that the location of an amino acid change in relation to the folded shape of the protein may be more important in affecting the chance of a shape change, rather than simply the number of amino acids that have been changed.

Hope for a cure

Tests for the presence of a disease organism can sometimes be based on a search for a particular amino acid sequence or protein fragment. Such tests would be of little use in this instance as both normal and afflicted individuals can carry the same sequence. Rather it is the presence of the dangerous or prion shape that must be identified. However, in the inherited forms of the disease (eg GSS) it would be possible to test for the sequences that appear more likely to spontaneously take on the dangerous shape. About 10 percent of prion diseases are familial in humans, killing half of the members of affected families.

Drugs could likely be developed that would bind to the safe form of the protein and prevent its shape being changed to the infectious form. Such a drug could delay or eliminate the onset of the diseases even in individuals carrying the prion form. Alternatively, it may be possible to knock out the prion form, perhaps using a vaccine that was specific to the prion shape. Due to the slow onset of the disease, a therapy that reduced the rate of onset may be sufficient to prevent the disease from developing to a damaging level within the natural term of life. In many countries the usual level of CJD is about one case (death) per one million people, however, post-mortem studies have suggested that as many as one in 10,000 humans actually have the disease, it is just that most die of something else before the condition develops.

There are some suggestions that other diseases that are not well understood (e.g., Alzheimers) may share some features in common with these conditions. Accordingly, further research into prions and protein shapes may cast light on other conditions and lead to new forms of control.

The recent media attention

The recent media focus on BSE and the risks of these diseases results from the discovery of ten individuals who contracted a spongiform disease whose onset occurred at an earlier age than was usually the case. The ten individuals had the normal sequence of amino acids in their form of the protein. It is possible that their deaths reflect contact with a variant prion that was more effective in changing the shape of their human protein than is normally the case in CJD.

A special investigation unit was set up in Edinburgh following the discovery of BSE in 1986, and only since this time has the death rate been carefully scrutinised. Some workers have suggested that these "new" cases are not new at all, rather no-one ever looked closely enough in the past and the deaths would have been attributed to other causes.

The recent decision to make CJD a notifiable disease in New Zealand will without doubt increase the apparent incidence of the disease even if there is no real change.

Risk of importing the disease

In general, there seems to be two schools of thought regarding importation of animal genetic material from countries affected by BSE and scrapie. The MAF viewpoint is to follow the world recommendations, with some additional safeguards. In the case of bovine material this allows semen or embryos to be imported from herds that have not ever had a case and have not been fed meals containing animal proteins. They would claim there is no evidence that the disease can be transmitted given these safeguards. In the case of sheep, the regulations are much stricter and long quarantine periods must be followed with embryos sourced from imported animals, washed a number of times, and all original animals slaughtered and examined. Some other views suggest that while there may be a very low risk there is certainly not zero risk. Accordingly, these workers would argue that importations should not occur under any circumstances.

Effects of these diseases

Some ill-informed individuals have suggested that New Zealand should capitalise on the British beef scare. These individuals have failed to recognise the limitations of market access to the European community and the moral issue of making the most of other peoples problems. The scare relating to consumption of British beef that the media have drummed up, will result in some substitution of British beef for other beef, and for other meats and protein sources such as lamb, chicken and meat substitutes. Although the current generation of meat-eaters will soon resume many of their previous consumption habits, the next generation of consumers will remember these fears and likely consume less meat than their parents.

Conclusion

The human diseases occur at very low frequency and have done so for generations. The animal conditions are not present in New Zealand. Links between BSE and CJD are possible but the evidence is not overwhelming. Research into prion diseases may prove of wider benefit to the understanding and treatment of other disease conditions.

Material in this issue of Breeding Matters may be reproduced provided the source is acknowledged.