1999 The Department of Animal Sciences Colorado State University

Bovine Spongiform Encephalopathy (BSE)

Henry N. Zerby and Keith E. Belk

On March 20, 1996, the Spongiform Encephalopathy Advisory Committee (SEAC) advised the government of Great Britain that the Committee had become concerned about 10 new cases of Creutzfeldt-Jakob disease (CJD). These cases were identified through national surveillance that between early 1994 and late 1995, detected an increase in younger people showing symptoms of CJD. The concern was that, in these new cases, humans may have contracted CJD by consuming food products that contained contaminated BSE beef. The basis of the Committee's concerns were:

• The new group of CJD patients was younger than those normally contracting the disease (new patients ranged in age from the late teens to early 40s; CJD usually has afflicted persons over the age of 63).
• A review of the new patients' medical histories, genetic analyses, and consideration of other possible causes of CJD failed to explain the cases adequately.
• Duration of illness lasted 13 months (normally it lasts 6 months).
• EEG electrical activity of the brain was not typical.
• Although the brain pathology was recognizable as CJD, patterns were different from normal CJD, with large aggregates of prion protein plaques (the brain pathology pattern was, however, totally different from that in the brains of BSE-affected cattle ).

SEAC concluded that, although there was no direct scientific evidence of a link between BSE and CJD, based on current data and in absence of any credible alternative, the most likely explanation at present was that these cases were linked to exposure to BSE before the introduction of control measures -- in particular, the ban on specified bovine offals (SBO). In 1989, the United Kingdom banned use of brain, spinal cord, tonsil, thymus, spleen, and intestines from cattle in food for animal and human consumption.

There is a lack of scientific evidence to support claims suggesting that a few cases of CJD in humans may be linked with exposure to food contaminated with BSE in Great Britain. The incidence of CJD in Great Britain has not increased significantly since the onset of the BSE epidemic in 1986, and remains similar to the incidence in other European countries. However, the media has promoted the story to epic proportions and has used sensationalism of the story to speculate and create skepticism concerning the safety of the beef throughout the European Union. Within days of the announcement of a possible BSE - CJD link, the European Union imposed a global ban on British beef exports. As a result, British and European Union officials agreed on a plan to begin slaughtering the British cattle herd to help re-establish consumer faith in British beef. A total of 4.7 million cattle over the age of 30 months, mostly dairy cattle, will be sent to slaughtering facilities. This will cost billions of dollars and cripple the beef industry in Great Britain.

What is BSE?

Bovine Spongiform Encephalopathy (BSE) is a member of a group of diseases known as Transmissible Spongiform Encephalopathies. These diseases cause neurodegenerative disorders in the brain and ultimately result in death. Bovine Spongiform Encephalopathy was first observed in Great Britain in April 1985. It was officially recognized as a new form of neurological disease in November 1986 by the Veterinary and Investigation Service and the Ministry's Central Veterinary Laboratory. Bovine Spongiform Enc ephalopathy is a lethal, slow-progressing, chronic-degenerative disease affecting the central nervous system of cattle. It is a fibril neurological disease that predominantly affects mature cattle and is characterized by the appearance of vacuoles in the brain of infected cattle, deteriorated neurons or clear holes that give the brain the appearance of a sponge.

Clinical signs of BSE include changes in temperament such as nervousness or aggression, abnormal stilted gait, high stepping, itching, excessive licking, and decreased milk production. Because of these clinical signs, BSE is more commonly known as "Mad Cow Disease". Since 1986, more than 158,000 cases of BSE have been identified in the United Kingdom, involving more than 50% of the dairy herds. It is common for only a few cattle from each herd to show clinical signs, rather than having large numbers in a single herd showing signs of infection (Prusiner 1993a). The Centers for Disease Epidemiology and Animal Health, USDA-APHIS-VS recently published a report that indicates 34% of farms with adult cattle in Great Britain have experienced at least one case of BSE (54% of all dairy farms and 15% of all beef farms). On affected farms, 36% have experienced only one case and 70% have experienced four or fewer cases.

About 98% of the cases of BSE have occurred in Great Britain. However, BSE has been reported in 10 countries outside the United Kingdom. In France, Portugal, Republic of Ireland and Switzerland, BSE occurred in native cattle; however, these cases were thought to be linked with importation of cattle feed from the United Kingdom. Identified cases of BSE in the Falkland Islands, Oman Sultanate, Germany, Canada, Italy and Denmark were in cattle imported from the United Kingdom. Most cases have been reported in the Holstein-Freisian breed, but all cattle are susceptible to BSE. The onset of clinical symptoms has been observed in cattle at an age of 22 months up to 15 years of age. Following the onset of clinical signs, the disease course varies from 2 weeks to 14 months. In that time, the animals' condition deteriorates until it dies or is destroyed. The incubation period of BSE ranges from 2 to 8 or more years.

Where did BSE come from?

There are different scientific theories concerning the origin of BSE. Epidemiological evidence suggests that the primary cause may have been feeding cattle rendered protein produced from carcasses of scrapie-infected sheep. Changes in rendering operations in the early 1980's--particularly the removal of a solvent extraction process (Prusiner, 1993b)--and the elimination of a second steam heat treatment may explain the appearance of the disease and a large increase in the number of cases subsequent to that time (Taylor, 1993). The Specified Bovine Offal Ban has led to a dramatic decline in new cases of BSE. This supports the hypothesis that feed-stuffs may have been a common infection source (Schreuder, 1994; Prusiner, 1993a). However, this theory has yet to be scientifically proven (Schreuder, 1994). Some evidence suggest that BSE may not be linked with scrapie because the scrapie agent inoculated into the brain of cattle does not produce BSE lesions (Cutlip et al., 1994).

Prion Protein

Bovine spongiform encephalopathy is caused by an unconventional infectious agent, originally described as a "slow virus" or as a "self replicating protein". More recently, it has been described as a "prion" - an aberrant conformation of normal protein (PrP c; Prusiner, 1993 a, b; Fairbairn et al., 1994). Several lines of evidence suggest that the prion protein is devoid of nucleic acid because, procedures known to damage nucleic acid (DNA and RNA) did not reduce infectivity (Weissmann et al., 1993). Prion proteins are encoded by a single exon (Weissmann et al., 1993) on a gene located on chromosomes (Fairbairn et al.,1994). Normal prion protein (PrPc) is synthesized in the endoplasmic reticulum, modified by the golgi apparatus and transported to the cell surface. The mature form of PrPc is bound to the outer membrane by a glycolipid anchor (Eissmann et al., 1993). A study conducted by Jeffery et al. (1995) indicates that prion protein is released from the surface of neurons, diffuses through the extra-cellular space, around infected cells, where it accumulates and finally becomes aggregated as amyloid fibrils. It is likely that the accumulation of the prion protein within the extra-cellular space is instrumental in causing nerve cell dysfunction and, ultimately, neurological disease (Jeffery et al., 1995). The function of the prion protein remains unknown (Fairbairn et al., 1994); it has been hypothesized to be involved with the synapses but clues from transgenic mice indicate that it is not necessary for normal development (Hope, 1995). Prusiner (1993b) has hypothesized that there are two forms of the prion protein. The normal form is referred to as "cellular PrP" (PrPc), and the variant form or the infectious (protease-resistant) form which is referred to as "scrapie PrP" (PrPSc). The main difference between the two forms is conformation or shape. Physical and chemical treatments that will normally inactivate conventional pathogens do not affect PrPSc (Fairbairn et al., 1994; Kingman, 1993). Even high-temperature autoclaving at 135'C for 18 minutes does not completely eliminate infectivity from contaminated tissues (Patterson and Dealler, 1995).

Increasing scientific evidence supports the hypothesis that prion molecules contact normal PrPc molecules in the membrane protein of the brain, and induce them to refold into the aberrant conformation, PrPSc. Molecules so formed go on to do the same to other normal PrPc molecules, thus creating further aberrant replicas from normal proteins (Kaneko et al., 1995). Normal prion protein PrPc and variant PrPSc have the same amino acid sequence. This is believed to be a major reason why the PrPSc prion protein does not cause an immune or inflammatory response as it is not viewed as a foreign substance by the body (Berg, 1994). Because the body recognizes PrPSc as PrPc, it does not realize that it is a harmful agent, so it does not defend itself from PrPSc . However, PrPc and PrPSc do differ in their conformational shape, or in the folding of the molecule. The normal PrPc form is made up of 42% alpha-helical structures and is practically devoid of beta-sheets, containing only 3%. In contrast the PrPSc c onformational shape is made up of 30% alpha-helical structures and 43% beta-sheets (Fairbairn et al., 1994; Cohen et al., 1994).

Replication

Replication of PrPSc is not completely understood. The current hypothesis -- with the most support -- suggests that PrPSc molecules act as templates. It is hypothesized that prion diseases result from a post-translational change in the conformation of PrPc to PrPSc (Lehmann and Harris, 1996). The PrPc contacts a normal PrPc and induces it to change its conformation and refold into the conformation that contains beta-sheets. Each one of these newly formed PrPSc molecules continues the process by contacting other PrPc, thus resulting in a "snowballing effect" that increases the number of PrPSc exponentially. Other theories on replication are being examined. Some researchers have hypothesized that a bacteria is responsible for transmitting the disease from animal to animal and that the bacteria causes the prion to replicate and fold incorrectly. Another hypothesis is that prions don't replicate; but, rather, that the prions accumulate in the brain from other areas of the body. Still others propose that BSE has a closer link to Alzheimer's disease than to CJD (Anthony and Parish, 1996). Little scientific evidence is available to support the latter theories.

Transmission

Only the brain, spinal cord and retina from naturally infected cattle have been found to be infected with the causative agent. In experimentally infected cattle, the brain, spinal cord, retina, and lower ileum have been found to be infected. Scrapie is apparently naturally transmitted between sheep; but there is no scientific evidence that scrapie can be transmitted to humans (Westaway et al., 1995). As with many issues surrounding BSE, the scientific literature does not agree and no conclusive evidence is available to support or refute the ability of BSE to pass horizontally (from cow to cow) or vertically (from mother to offspring) between animals. Bradley (1993) reported that there is no evidence to suggest that BSE is spread from infected cattle to non-infected cattle or from cattle to other species by contact. A report by the USDA Animal and Plant Health Inspection Service (1996) states that a case-control study found no evidence that maternal transmission occurred. Although there was evidence of marginally significant horizontal transmission, such a mode of transmission would not be capable of maintaining the epidemic. However, a report by Lacey (1996) suggested that there is reason to believe that vertical and horizontal transmission between cattle does exist. Dealler and Kent (1995) also indicated that vertical transmission may take place in utero .

Incubation periods for prion diseases are dependent on various factors. Levels of PrPc in the host, dosage or amount of exposure to contaminated source, route of administration, and genetic origin of contaminated source all affect the incubation period. Long incubation periods are related to what scientists have labeled as the species barrier (Fairbairn et al., 1994; Kocisko et al., 1995; Collinge et al., 1995). It appears that the species barrier between animals can be crossed in situations where infected material is injected into the brain of another animal. However, susceptibility to such a mode of transmission seems to be dependent on the similarity of the prion proteins between the two species (Collinge et al., 1995; Kocisko et al., 1995; et al., 1994).

Some prion diseases seem to be able to infect other species naturally while other species do not seem susceptible at all. Research has shown that transmission of a prion disease across species is likely to happen more readily when the amino acid sequence of prions in the two species are similar. PrP from the various spongiform encephalopathies has been sequenced and found to differ -- in some cases by very little and in some cases by quite a lot. Recent research has shown that the scrapie PrP protein differs from the BSE PrP protein at only seven amino acid locations, whereas the BSE PrP protein differs from the human CJD PrP at more than 30 locations. These differences explain the concept of strains and help explain why prions from one species might jump more easily into a particular species rather than another (Collinge et al., 1995).

Brandner et al. (1996) suggested that some regions of the prion protein may be more important for transmission than others, and that if the amino acid sequence in specific regions is homologous, it may explain how BSE could cross the species barrier into humans. However, to date there is no sound scientific evidence to support this hypothesis. Most scientific literature suggests that BSE is not able to cross the species barrier. In a study conducted by Hope (1995), mice that were genetically engineered to carry the human PrPc were subjected to the prion agent thought to be responsible for BSE. The agent failed to transmit its effects on the human PrPc. The latter study further indicated that humans can not contract neurodegenerative diseases by eating beef products contaminated with BSE. This has caused many people to be skeptical of BSE's ability to cross the species barrier. However, there is strong evidence suggesting that cats can contract neurodegenerative diseases by consuming beef infected with BSE (Patterson and Dealler, 1995).

Confirmation of BSE in cattle is only possible following postmortem examination of the brain tissue. This is partially because the mutated prion does not produce a detectable immune response; so, there are no immunological or serological diagnostic tests available to identify cattle infected with BSE (Schreuder, 1994; Berg, 1994). Several methods for live animal testing are presently being investigated and a spinal fluid test may soon be released.

Cure and Prevention

No cure has been developed to date and it is not likely that one will soon be found. However, some preventive measures are being researched. The first, explained by Westaway et al. (1995), involves selecting livestock in which the PrP-encoding gene has been deleted (assuming that omitting the prion protein does not impair desirable characteristics). A study conducted by Brandner et al. (1996) showed that PrPc must be present for PrPSc to have a detrimental effect on the animal. Eliminating their presence eliminates the mode of replication. Other methods of prevention entail the development of a compound that can bind to PrPc and stabilize the conformational shape, thus preventing the conversion of alpha-helices to beta-sheets.

Other Transmissible Spongiform Encephalopathies (TSEs)

Transmissible spongiform encephalopathies are naturally occurring diseases in some mammals. There are known types in humans and in animals.

• Scrapie:
  Found in many parts of the world, Scrapie was recognized as a distinct disorder of sheep as early as 1738 (Prusiner, 1993b). Work in Iceland showed that pastures where infected sheep have grazed may retain the condition that causes Scrapie and infect unrelated sheep at a later time when grazed on the same pasture (Dealler and Lacey, 1992). Some breeds seem to be more resistant to Scrapie than are others. Scrapie appears to be transmissible maternally through the placenta.
  Most often sheep on farms that are found to be infected with scrapie are more than five years of age. Sheep showed increased irritability, excitability and restlessness at the onset, giving rise to scratching, biting and rubbing the skin (hence the name scrapie). They will also show loss of weight, weakness of the hindquarters and -- in some animals -- impaired vision. The disease is always fatal. Only a small number of animals in each herd suffer from the clinical signs of Scrapie; rarely are there 10 cases within the same herd. Scrapie does not appear to be able to infect humans.
  
  
• Kuru:
  Kuru is associated with the Fore tribe of the Okapa district of the Eastern Highland in Papua, New Guinea. Kuru was reported in 1956 and appeared to be spread by the ritualistic practice of cannibalism (Dealler and Lacey, 1992). In 1960, cannibalism was abandoned and the incidence of kuru dropped dramatically in subsequent years. Clinically, the disease is a progressive brain disorder (cerebellar ataxia) leading to uncoordinated movements, neurological weakness, and decay in brain cortical function. Most patients that die of kuru are not demented. This is the major clinical difference between Kuru and CJD. Most deaths reported were among women and children as they consumed a mash consisting of the brains of deceased humans while men in the tribe consumed the meat (Dealler and Lacey, 1992). The Kuru causative agent can remain quiescent for up to three decades (Prusiner, 1993b).
  

• Creutzfeldt-Jakob Disease (CJD)
  Creutzfeldt-Jakob Disease was first described in 1920 and 1921 when it was known as a "spastic pseudosclerosis", or "subacute spongiform encephalopathy" (Dealler and Lacey, 1992). The illness exists throughout the world and has a similar prevalence in each of the countries tested --with an annual incidence of approximately one case per million inhabitants (Dealler and Lacey, 1992; Prusiner, 1993). There are three forms of CJD that are recognized: (1) infectious, (2) sporadic, and (3) inherited. The only documented cases of infectious CJD are iatrogenic (passed to a patient through medical procedures), where accidental transmission of CJD to humans has occurred with corneal transplants, growth hormone injections, or with surgical procedures that involve d infected instruments. In recent years it has also been shown that CJD can be transmitted to humans by treatments that involve administration of natural human growth hormone or grafting of tissue surrounding the brain. However, there is no evidence that supports the possibility of contracting CJD from normal human to human contact-routes other than the iatrogenic model (Hainfeller et al., 1996). The majority of CJD cases are sporadic and 10 - 15% of CJD cases are familial, inherited as an autosomal dominant trait (Prusiner, 1993). Epidemiological studies have failed to implicate the ingestion of scrapie-infested sheep meat or goat meat in the pathogenesis of CJD in humans. However, speculation regarding this possible route of infection continues.
  Clinical symptoms of CJD start with changes in sleeping and eating patterns and progress over a few weeks into a clearly neurological syndrome. A rapid onset of neurological symptoms appears in 20% of the cases -- most commonly those of vibrating muscular spasms, dementia, loss of higher brain function, and behavioral abnormalities. The disease progresses with deterioration in cerebral and cerebellar function, to a condition in which most neurological activity is decreased, sensory and visual function decays, and the patient dies -- possibly after a decrease in the lower motor neurological function and seizures. The majority of the cases of CJD occur in older patients. Ninety percent of the cases end in death within one year. However, for 5% of the cases, fatality may take up to 10 years. In these cases, neurological decay is relatively slow.
  
  
• Gertsmann-Straussler-Scheinker Syndrome (GSS):
  First described in 1936, most cases of GSS are familial, exhibiting an autosomal dominant pattern, but sporadic cases resembling GSS clinically and pathologically have been reported (Prusiner, 1993b). The disease is similar to CJD except that it has a more extended onset and duration, a tendency toward cerebellar ataxia as the initial predominant neurological sign, and a large number of amyloid plaques present among the spongiform encephalopathic changes of the brain (Dealler and Lacey, 1992).
  
  
• Alper's Disease:
  Alper's Disease represents a group of very rare, chronic and progressive degenerative disorders of the central nervous system of infants and children. Histologically, this condition is similar to CJD; however, there is also a fatty degeneration of cirrhosis of the liver (Dealler and Lacey, 1992).
  
  
• Transmissible Mink Encephalopathy (TME):
  Transmissible mink encephalopathy is an uncommon fatal disease that occurs as outbreaks in ranch mink. The condition was first reported in 1947 in Wisconsin and has also been reported in Canada and Finland with a similar pattern (Marsh and Bessen, 1993). Most of the mink on a farm die rapidly after a short encephalopathic period. The approximate incubation period is six months. The disease is thought to be derived from food which is contaminated with a transmissible spongiform encephalopathy of another animal. Suspect sources include scrapie-infected sheep and goats and BSE-infected cattle (Marsh and Besson, 1993; Westaway et al., 1995).
  
  
• Chronic Wasting Disease:
  Chronic wasting disease of deer is a transmissible spongiform encephalopathy first seen in 1978 in a mule deer herd and in an adjacent herd of elk in northern Colorado. Both herds were captive. The disease shows a typical spongiform change in the cerebral grey-matter (Dealler and Lacey, 1992).
  
  
• Feline Spongiform Encephalopathy (FSE):
  Feline spongiform encephalopathy is a condition that was reported in May 1990 in a five year old male Siamese cat and has since been reported in other cats in the United Kingdom as well as a puma and a cheetah. It is believed that the animals contracted the disease from consuming food that was contaminated with the causative agent of BSE before the specified bovine offals ban (Dealler and Lacey, 1992; Schreuder, 1994). Evidence that domestic cats have contracted FSE from a food source associated with BSE is also supported by a report by Patterson and Dealler (1995).
  
  
• Zoological Spongiform Encephalopathy or Exotic Ungulate Encephalopaty:
  Zoological animal transmissible spongiform encephalopathy has been reported since 1986 in an eland, a nyala, an oryx, a kudu, and a gemsbok in British zoos. These animals became clinically unwell after the appearance of BSE on British farms, and are suspected to have been infected either from the same source as cattle or from BSE-infected food or feed (Dealler and Lacey, 1992).

Summary

Unfortunately, there are no real answers to the questions surrounding BSE. There is an extensive amount of research being conducted presently, but the scientific investigations concerning prion diseases are still in their infant stages. Little is understood about the mechanisms associated with PrPSc replication and the pathology, transmission and replication of prion related diseases.

There is an enormous amount of scientific literature that clearly disputes any connection between scrapie in sheep and CJD in humans. Similar claims have been made disputing any link between BSE and CJD, but the fact that BSE can infect cats, most likely by the oral route (which for other spongiform encephalopathies is not the most effective route) has led to concern among the general public in the United Kingdom. Even as a majority of the evidence in the scientific literature seems to suggest that BSE does not impose a health risk in the human food supply, precautionary measures should be employed until more evidence can be gathered. A lesson can be learned from past situations, such as occurred during early stages of the AIDS epidemic. In the case of AIDS, some actions reported to be safe were later found to impose a risk of infection. Most experts acknowledge that there is no definitive evidence to either support or refute the theory that CJD can be caused by exposure to BSE.

Relative to the U.S. beef industry, the major concern is: Are any cattle in North America infected with BSE? The U.S. Department of Agriculture has declared that there have been no cases of BSE in the United States. In 1986, USDA established a comprehensive BSE surveillance program involving more than 60 diagnostic laboratories. Over the past 10 years, the USDA's Animal and Plant Health Inspection Service (APHIS) has tested 2,791 cattle that exhibited traits even remotely similar to traits associated with BSE. None of those animals tested positive for BSE.

Since 1989, APHIS also has enforced a ban on the importation of live ruminant animals and of ruminant-derived products from countries where BSE is known to exist. On March 29, 1996, USDA announced additional layers of protection including expanded surveillance, new regulations that will make the current voluntary ban on feeding ruminant-derived proteins to ruminants mandatory, and increased industry awareness and educational programs to further protect against the disease.

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