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Prions and BSE – Mad Cow Disease

In 1986, several U.K. cattle came down with a new illness, promptly named Mad Cow Disease because of its neurological symptoms. Within a few years, Mad Cow Disease or B.S.E. was epidemic in British cattle and was determined to be caused by feeding cattle food supplements made from sheep remains that were infected with a similar form of the disease. In March, 1996, the British government announced that there was a possible link between a new human disease variant called Creutzfeld-Jakob disease (vC.J.D.) and the ingestion of beef from cows infected with B.S.E.. The British beef market fell by 30 percent within two days. As of January, 2004, there have been 153 reported cases of vCJD worldwide. 143 of these cases were located in Britain. Other cases have been reported in France, Canada, Ireland, Italy and the U.S.. Because of the relatively long incubation period of the disease, it is possible that more cases will develop. Is this the beginning of a new human epidemic? Or has the disease peaked and a decline in cases observed as the many new regulations and precautions take effect?

B.S.E. is an acronym for Bovine Spongiform Encephalopathy. B.S.E. has a very long incubation period of four to five years. It is always fatal causing death within weeks to months of symptom onset. The brain of a deceased cow infected with this disease appears ‘sponge-like” as it has many small holes in the tissue caused by the death of many brain cells or neurons. As the brain tissue slowly dies, the animal loses control over its movements and behavior – appearing “mad”. During the final stages, infected animals become aggressive, lack co-ordination, and are unsteady on their hooves.

Both cows and sheep are naturally plant-eaters. This has been true until modern agricultural practices were developed which involved the feeding of protein supplements containing the rendered body parts of other animals. The animal protein did encourage more rapid growth of the cattle, but also opened a new route of transmission for the infectious agent that causes B.S.E.. The outbreak of BSE in the UK occurred because of the practice of feeding cattle protein rendered from the remains, including brain matter, of scrapie-infected sheep. The disease known as “Scrapie” has been present in British sheep for at least 200 years. The name “Scrapie” was coined by Scottish shepherds to describe the fact that infected sheep would scrape themselves against trees because of the itching sensation caused by the disease. It was determined that the infectious agent causing Scrapie was the same as the one causing B.S.E..

The problem was then compounded by feeding cattle protein rendered from other infected cow carcasses. The mad cow epidemic reached its height in the 1980’s having infected more than 180,000 British cattle.

Scrapie is the original example of a group of diseases known as the “Transmissible Spongiform Encephalopathies” (T.S.E.). These diseases are also known as the “prion” diseases and include Scrapie, B.S.E., and vCJD. Other known human T.S.E.’s are Famileal Fatal Insomnia (FFI), Kuru, and Gerstmann-Straussler-Scheinker disease (GSS). Other animal T.S.E.’s include Feline Spongiform Encephalophy (FSE) in cats and Chronic Wasting Disease (CWD) in deer.

T.S.E.’s or prion diseases all affect the brain and destroy neuron cells in large numbers creating holes or microlacunae in the brain tissue which are visible with an ordinary microscope. The holes are filled with amyloid-like deposits.

The term “prion” was first used in 1981 by Dr. Stanley Prusiner at the University of California to identify the agent that causes TSE’s. The cause of these diseases was suspected to be viral until the 1960’s when T. Alper suggested that the infectious agent that causes “scrapie” might have the ability to replicate without nucleic acid. All known viruses have a nucleic acid component. In 1967, J.S. Griffith proposed the Protein Only Theory to explain how prions could replicate if they were composed only of protein. This was (and still is) controversial as no life form including viruses had yet been found that did not have nucleic acid as the encoder of the chemical information used for reproduction.

Normal animal cells make prion protein which is known as cellular prion protein (PrPC). Scientists are not sure what the function of this normal prion protein is. It is possible that these shape-changing proteins are involved in the formation of memories. All mammals have prion protein genes. The gene sequences are similar in different species, but are not identical. In T.S.E., abnormal prion protein is present which can convert normal cellular prion protein to the abnormal form. For example, in Scrapie the abnormal protein is known as PrPSc. PrPSc converts PrPC to PrPSc. How does it do this?

Proteins are chains of chemicals called amino acids that are linked together like beads on a string. The prion protein has about 250 amino acids. Proteins do not remain linear as the properties of the different amino acids make the protein fold up into a particular shape or conformation. A protein’s conformation is a factor in determining it’s biological properties and function. The theory of Protein Only replication is that the abnormal prion PrPSc can convert normal cellular prions to abnormal prions by “flipping” their shape or conformation. These flipped rogue prions can go on to infect other cells or animals. A chain reaction occurs where the newly converted proteins convert other proteins with which they come into contact. This conformational conversion has been experimentally observed at the cell membrane surface of neuron cells in the laboratory.

Following ingestion or inoculation, PrPSc may accumulate in the lymphoid tissue of an animal even if the animal’s immune system is intact. The invading PrPSc flips normal cellular PrPC at the cell membrane surface. The PrPC in a cell continues to be flipped and the resulting PrPSc accumulates in small vesicles in the cell. It is theorized that the prion filled vesicles could enter the brain by flowing along the axons of neurons. The PrPSc prion responsible for Scrapie has been shown to possess two highly hydrophobic regions capable of spanning the cell membrane of neurons and disrupting them causing cell death.

Many experiments have been performed over many years that look directly or indirectly for nucleic acids in TSE infectious material. None has been found. Infectious material has been treated with chemical, irradiation, and enzymes which destroy any nucleic acid that might be present – but the material still remains infectious.

Another theory suggests that nucleic acids are a factor in the infectivity of prions. There are many different kinds or “strains” of TSE. These are differentiated from each other by the incubation period and the amount of lesions in the brains of mice when purposely infected. Fifteen different strains have been identified. Since a lot of information is needed to produce these different strains, it is theorized that nucleic acids are involved. It is also possible that host-encoded stimulatory RNA molecules may play a role.

Varient Creutzfeld-Jakob disease, vCJD, the major form of TSE affecting humans, was first described in March 1996. Traditional CJD in humans is rare and is thought to be a genetic disease. vCJD affects younger patients (average age – 29 years), has a longer duration (median of 14 months), and is strongly linked to exposure through food to BSE. vCJD starts out with psychiatric symptoms often characterized as depression or a schizophrenia-like psychosis. Unusual sensory symptoms such as “skin stickiness” have been reported. As the disease progresses, further neurological problems develop such as unsteadiness, difficulty walking and involuntary movements. The patient is completely immobile and mute by the time of death. Confirmation of the diagnosis of vCJD is currently only possible by post-mortem examination of the brain. Under microscopic examination, multiple abnormal aggregates called “florid plaques” encircled by holes are observed in the brain specimen.

The human disease “Kuru” first appeared in the early 1900’s in New Guinea. It reached epidemic proportions in the 1950’s among the natives of the South Fore region of the country. Over 1,100 people died of the disease between 1957 and 1968. Most of these cases were women. Anthropologists determined that the cause was the ritual acts of mortuary cannibalism which was practiced in the area. Upon the death of an individual, the women would dismember the corpse, strip the limbs of muscle, and remove the brains and internal organs. The organs were lightly cooked and eaten or smeared on the bodies of the funeral participants. After this practice ceased, the incidence of Kuru decreased to almost non-existent levels. The symptoms of Kuru are similar to vCJD – depression, tremors, unsteadiness, inappropriate laughter, and ultimately death. Kuru was first thought to be genetic, then viral and then a prion disease after Prusiner identified and defined TSE’s in the 1980’s.

No infectivity has yet been detected in skeletal muscle tissue. Milk and milk products are considered safe. Tallow and gelatin are also considered safe if they have been prepared by a process that inactivates prions and has been prepared from specifically identified tissues or from cattle without risk of BSE exposure. The pharmaceutical and cosmetics industries need to take precautions as well in their use of raw materials made from bovine and other potential TSE species. Both human and veterinary vaccines prepared from bovine tissues may carry the risk of TSE transmission. Prions are highly resistant to inactivation by freezing, drying, and heating at normal cooking temperatures, even those used for pasteurization and sterilization. They are also highly resistant to UV irradiation and strong degradative enzymes. Prions can be inactivated by autoclaving dry waste at 132 degrees C. for 4.5 hours. Large volumes of wet waste can be treated by treating with 1N NaOH followed by autoclaving at 132 degrees C for 4.5 hours. Prions can also be inactivated with concentrated solutions of disinfectants like phenol and sodium hypochlorite for 24 hours.

Evidence that there is a link between vCJD and BSE comes from the observance of pathological features similar to vCJD in Macaque Monkeys inoculated with BSE. In order for people to be infected with PrPSc prions, they must eat nervous tissue from an infected animal. In the past, this was possible through processed foods such as sausages.

On December 22, 2003, testing showed that an American cow slaughtered in Washington was infected with BSE. Countries around the world promptly banned American beef with serious economic consequences. It has been confirmed that the cow had been imported from Canada. Large quantities of cattle feed had been imported from the UK to Canada during the peak years of the mad cow epidemic, and it is believed that the American cow as well as two other confirmed Canadian cows infections were a result of this infected feed.

Because of the mad cow finding in Washington, the U.S. Department of Agriculture initiated new regulations aimed at preventing BSE-infected material from entering consumer meat products.

1) “Downer Animals” are banned from the food chain. A Downer is an animal that cannot stand up on its own.

2) BSE testing within the food chain has been made more frequent and rigorous.

3) The following specified risk material has been prohibited from entering the human food chain – skull, brain, trigeminal ganglia, eyes, vertebral column, spinal cord, dorsal root ganglia of cattle over 30 months of age as well as the small intestine of cattle of all ages.

4) The practice of “Air Injection Stunning” where an air-driven bolt is driven into a cow’s brain during slaughter. With this method there is a possibility that brain matter could be driven into the circulatory system.

5) The practice of mechanical meat separation – a process where machines strip the last small remnants of meat from cattle carcasses has been prohibited. This practice could pick up bits of brain and spinal material.

Much research is underway to further clarify the biochemical nature and infection pathways of the prion protein and it’s relationship to the TSE diseases. This research is also attempting to find cures for the prion diseases – especially variant Creutzfeld – Jakob disease. There have been some positive laboratory results with antibodies and drugs (pentosan polysulphate). The antibodies may work by blocking the normal version of the prion protein (PrPC) from being produced on the surface of cells. This in turn would block the contact of the normal proteins with the abnormal prion protein (PrPSC) which is necessary for conversion and entry into the cell. The Pentosan drug may work in two ways – after injection into the brain, it should stop production of the abnormal prion protein, and secondly, it could cut the activity of human inflammatory molecules called interleukins. Interleukins are stimulated by abnormal prions and can then attack and destroy brain cells.

The BSE epidemic reached its peak in January 2003 at approximately 1000 new cases per week. It appears that the precautions taken, beginning in 1994, to reduce the risk of eating infected beef products has been effective. It is estimated that the median incubation time for vCJD in humans is 7.4 years. Human vCJD cases reached a peak in 2002 with 93 human infections. In 2003 there were 83 cases. 40 cases are projected for 2004. The predicted deaths from vCJD have been revised downward substantially. In 1997, it was predicted that up to 10 million people could die from this terrible disease. In 2002 the figure dropped to 50,000. In February, 2003, the likely upper limit of deaths has fallen to a maximum of 7000 deaths and a minimum of 80 deaths from vCJD by the year 2080 (95% confidence limits).

In conclusion, there is much still yet to be discovered about this strange, infectious agent known as a prion. A whole new world has been discovered whereby biological replication appears to be possible without the requirement for nucleic acid involvement. Cross-species infection has been proven, most seriously between cattle and man through the consumption of infected beef products. The good news is that actions and regulations that have been enacted for BSE containment appear to have been effective in reducing the incidence of this disease.

Sources:

1. Destruction Of Select Agents Procedures

www.ehrs.upenn.edu/protocols/sa_destruct.html

2. Mad Cow Disease Images & BSE Pictures

www.rkm.com.au/BSE/

3. World Health Organization. Bovine Spongiform Encephalopathy

www.who.int/mediacentre/factsheets/fs113/en

4. The Environmental Literacy Council – Mad Cow Disease

www.enviroliteracy.org/subcatagory.php/153.html

5. Kuru: The Dynamics of a Prion Disease

www.as.va.edu/ant/bindon/ant570/Papers/McGrath/McGrath.htm

6. World Health Organization. Variant Creutzfeldt-Jacob Disease