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Back To Vidyya Should We Fear The Cow?

Bovine Spongiform Encephalopathy And Creutzfeldt-Jakob Disease

In the spring 1996, an invariably fatal degenerative brain disease appeared among several young people in Great Britain. Although its causative agent has not yet been identified irrefutably, it was feared that the victims had been eating or handling beef products contaminated in some way, and that the humans contracted the disease from the beef. Later that year, in what some might describe as a panic-inspired move, the British government ordered the slaughter and incineration of more than 37,000 head of cattle. Almost immediately thereafter, other European countries banned the import of British beef, dealing that country's beef industry a staggering economic blow. The United States had already banned beef from Great Britain in 1989.

To add to this economic woe, in July 1997, the European Union banned orally ingestible medicines that contain animal by-products; the ban took effect 1 January 1998. About 80% of such medicines contain tallow or gelatinous material made from animal sources, largely from cattle. The FDA opposed the ban fearing it would lead to severe shortages of orally ingestible medicines worldwide. Citing potential shortages and the continuing need for the medicines, FDA officials put pressure on the European Union to hold back on the ban. The US pharmaceutical industry alone, stood to lose $4 billion in annual exports.

One reason for these actions is that many bovines in Great Britain were stricken with bovine spongiform encephalopathy (BSE), or mad cow disease. In this sickness, the disease agent eats spongy holes in bovine brains, making bulls and cows behave strangely and eventually killing the animals.

The most compelling reason, however, likely was the appearance among humans of a new variant of the rare but dreaded Creutzfeldt-Jakob disease (CJD), a human spongiform encephalopathy believed to have been transmitted to the patients from contaminated beef products (however, the means or route of infection is still not known). Worse, this variant, known as vCJD or nvCJD, tends to afflict relatively younger people, even adolescents; normally, CJD very rarely strikes anyone under the age of 45. Moreover, the incubation period of vCJD appears to be only a few years, compared with the several decades believed to be required for normal sporadic or familial CJD. Researchers in this field note, however, that the incubation time and pathological manifestation of the disease is influenced by factors such as the route of inoculation, the dose and source of the infecting agent, and the genetics of the host being infected.

Whether normal or variant, CJD is a rare, yet horrible, disease that normally strikes 1-2 persons per million and is always fatal, generally within one year. Its outward symptoms in humans resemble those of Alzheimer's disease in several ways: depression sets in, dementia begins and then accelerates, and eventually the patient loses all physical control of his or her functions. However, necropsies of brains of Alzheimer's and CJD patients show a significant difference: brains of Alzheimer's patients are laced with amyloid plaque, whereas those of CJD patients, while also containing amyloid plaque deposits, are found to be full of spongy holes, principally in the cerebral tissue. Because of these spongy holes, CJD and its equivalent in animals is also called spongiform encephalopathy (SE).

As of December 1997, at least 25 people in the United Kingdom and France had contracted vCJD. Each of these victims is thought to have eaten or worked with contaminated beef or beef products. Results of recent research indicate compellingly that vCJD and certain types of SE are transmissible among animal species, hence the name transmissible spongiform encephalopathy (TSE). Moreover, TSE, believed to have been transmitted via ingestion of cattle remains, has appeared in domestic and zoo felines. Using infected tissue from cattle and from necropsies of vCJD patients, researchers have induced TSE in mice and macaque monkeys. However, the only SE known to be transmitted from animals to humans has been BSE, and the means of transmission remains unknown. It should be emphasized that, while many researchers maintain that no transmissible disease agent has so far been identified irrefutably, there are strong indications of SE transmissibility.

Did BSE Begin in the Feedlot?

BSE began to show up in cows several years after cattle feed manufacturers, mainly in Great Britain, changed the way in which waste remains from sheep slaughter, consisting partially of brain and nervous tissue, eyes, spleen, stomach, and other organs, were prepared for cattle feed. In the late 1970s, the hydrocarbon-solvent extraction method used in the rendering of offal began to be abandoned, resulting in meat and bone meal with a much higher fat content. In the 16 September 1997 issue of Proceedings of the National Academy of Sciences , Stanley B. Prusiner, a professor of neurology at the University of California at San Francisco, and colleagues suggested that this change in the rendering process had allowed the SE-causing agent from sheep to survive rendering and to be passed to cattle by a path still not clearly understood.

Brain tissue
Brain tissue

Potholes in the brain . Histological stains show vacuolization of brain tissue in a BSE-infected cow (left) and a human with CJD (right).

BSE was reported in the United Kingdom in the early 1980s and by 1987, there were 446 cases. The practice of feeding the remains of sheep and other ruminant animals to cattle was banned in 1989; however, the number of cases of BSE reported increased steadily until 1992, when it peaked at 36,682 cases. Since then, the number of reported cases of BSE in the United Kingdom decreased to 8,150 in 1996, and 2,858 through 31 October 1997. In 1999, the last year in which records are readily available over 2,000 cases were still detected. It is estimated that a total of 1 million cattle were affected by BSE, assuming an incubation period of five years. Prusiner and his colleagues consider this figure an underestimate, given that many cattle, afflicted or not, normally were slaughtered at the age of 2-3 years. Nevertheless, during the past decade, at least 160,000 head of cattle have died of BSE. Human CJD, incidentally, was designated by British authorities as a reportable disease in 1989.

Currently, it appears that the trend of new cases of BSE in the United Kingdom and in several other countries where BSE has appeared (though in the dozens of cases, at most, unlike the thousands reported in the United Kingdom) is declining. Preliminary results released by the United Kingdom Ministry of Agriculture, Fisheries and Food, however, suggest that BSE can be passed from a cow to her calf, which might conceivably lengthen the time needed until BSE is eradicated. Nevertheless, maternal transmission rates were put at about 10%; further evidence suggests that only cows in the last six months of infection pass BSE on to their calves. But the study did not make clear whether maternal transmission occurred in utero , at birth, or soon after birth, according to the United Kingdom's Spongiform Encephalopathy Advisory Committee. Moreover, no infectivity had been reported in infected cows' placentas, blood, or milk as of August 1996.

How did BSE come to affect cattle in the first place? Science has long known about ovine spongiform encephalopathy, or OSE (shepherds call the disease "scrapie," because in addition to becoming listless and behaving strangely, as do BSE-affected cattle, sheep scrape themselves almost incessantly against poles, stakes, fences, trees, or rocks until they lose much of their fleece and even sizable chunks of flesh). Thus one might wonder why, during the late 1970s, no one reminded the scientific community and health authorities that sheep farmers had known about OSE for at least the previous 200 years. Moreover, according to an article in the 10 August 1996 issue of Lancet , vertical transmission of OSE (mother to offspring) is generally accepted as being much more common among sheep than is vertical transmission of BSE in cattle.

Since BSE and vCJD appeared, researchers in the field have been unable to agree on what the causative agent that could act on different animal species might be. In a report drafted on 12 January 1997, the U.S. Centers for Disease Control and Prevention suggests, "The etiologic agent is thought to be an unconventional filterable agent. Currently, there is no test to detect a patient's immunologic response to the infection and the etiologic agent has not been identified. The exact mode of transmission in humans is not known; however, the disease can be induced in laboratory animals by percutaneous inoculation of infective material (brain tissue or cerebral spinal fluid from an infected person). Transmission in iatrogenic cases has been associated with the use of contaminated instruments (i.e., neurosurgical instruments or stereotactic electroencephalogram or cortical electrodes used on infected brain tissue, corneal transplants, and dura mater grafts), transplantation of brain tissue, or inoculation of contaminated human pituitary derived growth hormone. Person-to-person transmission via skin contact or via environmental contamination has not been shown."

The CDC's comments notwithstanding, the 1997 Nobel Prize for Medicine was awarded to Prusiner for having identified rogue proteins, known as prion proteins (PrP), that have gone awry in the brain as the proximate cause of SE. That year's award was unusual in that no other researcher shared the prize and that the Karolinska Institute in Stockholm, Sweden, which awards the Nobel Prize, gave it for such cutting-edge work. According to reports in the scientific and popular press, the prize committee characterized Prusiner's discovery of prions as the finding of a new biological principle of infection.

Nevertheless, several scientists differ with this finding. For example, Alan Ebringer of the Infection and Immunity Group in London proposed in the November 1997 issue of EHP that BSE is a form of autoimmune disease. Laura Manuelidis, head of neuropathology at Yale Medical School in New Haven, Connecticut, believes a virus will turn out to be the SE culprit. Frank O. Bastian, director of neuropathology at the University of South Alabama in Mobile, posits that the cause is a Spiroplasma , a microorganism that lacks a cell wall. Robert Rohwer, director of the neurovirology unit at the Veterans Affairs Medical Center in Baltimore, Maryland, was quoted in the 12 July 1996 issue of Science as characterizing the prion hypothesis as the cold fusion of infectious disease; he called for a very high level of skepticism and scrutiny before it's adopted. Many who differ with the prion theory maintain that the concept of a protein acting alone as a cause of infection and replicating without DNA, RNA, or both is at odds with generally accepted principles of biology.

Doubts about the Smoking Gun

Essentially, the prion hypothesis states that rod-shaped prion proteins, which exist throughout human and animal brains, cause SE when they mutate into fibril form and begin eating holes among the neurons. A gene in the brain apparently encodes the formation of PrPs. Normal prions are known as PrP C and are harmless (their function is not yet understood), whereas the mutated, and presumably SE-causing variety is designated as PrP Sc . According to one concept, a mutated prion-coding gene codes for PrP Sc generation. Then, interaction between PrP C and PrP Sc molecules somehow causes the normal form to change to the infective form and attack neurons. The newly formed PrP Sc then corrupts another PrP C , and so the chain continues, a sort of gruesome biochemical analogue of Gresham's law of economics, which holds that bad money chases out good. Another view is that, once formed, the PrP Sc molecule joins with a PrP C molecule to form a PrP Sc seed; next, infection by PrP Sc begins a fast polymerization of these rogue molecules, which might be regarded as a kind of self-replication.

Apparently, PrP Sc is an isomeric form of PrP C with altered chemical properties. For example, PrP C can be destroyed by protease enzyme, but PrP Sc cannot. Those who espouse the prion hypothesis believe PrP Sc to afflict all human and animal brains with SE, be it sporadic and familial CJD, vCJD, BSE, or scrapie.

So despite studies in which BSE and vCJD were shown to be transmitted to mice and macaque monkeys, researchers have yet to dispel all doubt that a rogue PrP is the smoking gun. What would be needed as a first step toward proof is a synthesis of normal PrP--PrP C --that can be rigorously certified as free of nucleic acid. The next step would be to demonstrate that the PrP C can change into the PrP Sc state and infect animals. As of mid-1996, no one had been able to create a prion, much less have it transmute and cause infection.

Some Suggested Alternatives to Prions

Considering possible causes of SE, some researchers ask, is the alternative something other than prions, or is it something that must be present to cause normal prions to go awry?

An autoimmune disease . Ebringer and his colleagues proposed in their EHP article that BSE is caused by cross-reactive antibodies evoked following exposure of cows to biological material from cows and sheep containing bacteria that may cross-react with bovine self-antigens. He names an Acinetobacter , an Agrobacterium , and a Ruminococcus as such bacteria, because of a resemblance of some of their protein sequences to that of bovine myelin.

Following exposure, damage to nerve tissue takes place in two stages, according to Ebringer. First, the outer covering of neurons--the myelin sheath--is damaged, thereby exposing nerve tissue. Then, damage to neurons occurs with relative accumulation of self-proteins, such as PrP, that cannot be readily hydrolyzed. Ebringer says that injection of infected brain tissue into experimental animals causes a neurological immune disorder called experimental allergic encephalomyelitis (EAE), in which the destruction of myelin brings about vacuolization (sponginess) with subsequent destruction of neuronal tissues. As a culprit, he suggests the molecular mimicry between bacteria and brain tissue, which does not conflict with existing tenets of biology. He proposes testing this hypothesis by examining sera from BSE-afflicted cattle for antibodies to these bacteria.

Prions Puzzling prions . Scientists are creating three-dimensional images of prions, such as the recombinant Syrian hamster prion protein, to solve the mysteries of BSE and related diseases. Source: Molecules R Us home page, National Institutes of Health, at From: James TL, Liu H, Ulyanov NB, Farr-Jones S, Zhang H, Donne DG, Kaneko K, Groth D, Mehlhorn I, Prusiner SB, Cohen FE. Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform. Proc Natl Acad Sci USA 94:10086 (1997).

An elusive virus . Manuelidis questions the concept of rogue prions as the cause of SE and blames the disease on a virus. A virus associated with SE has not been found so far, but Manuelidis observes that it took hundreds of researchers 10 years to find the hepatitis C virus. In addition, she reported in 1989 that she separated different molecules in infectious brain samples and found that the most infectious material did not contain the most PrPSc (other laboratories have reproduced this finding). Rather, its density was between that of protein and a nucleic acid, which one would expect of a virus, according to Manuelidis.

Spiroplasma . Bastian rejects the hypothesis of prions as the cause of TSE. Instead, he views PrP Sc as a disease product, rather than a source of the infection.

Discovered in 1976, Spiroplasma is an organism that not only has no cell wall, but also blends with the cell membranes of its host. Bastian told the National Managed Health Care Congress's Second International Conference on Transmissible Spongiform Encephalopathies, held in Washington, DC, in November 1997, that he has detected genetic material from Spiroplasma , particularly Spiroplasma mirum , by polymerase chain reaction (PCR) in 11 cases of CJD, but not in 10 controls. Direct sequence of the PCR product shows 98.5% homology with Spiroplasma DNA, according to Bastian. He also has shown the Spiroplasma gene in scrapie-infected hamster brains. Bastian presented evidence that the immune system is involved in TSE, and that this agent develops or replicates in a state of immune-tolerance.

In addition, the causative agent is hard to destroy. It seems to remain virulent after it is boiled in water or run through a rendering process at the temperatures of melted animal fat. At the TSE conference, Bastian pointed out that the agent is not destroyed at pH 2-11; he has shown evidence that Spiroplasma survives this wide range of pH shifts. He speculated that survival through the stomach would be more characteristic of a living organism than of a protein, which would tend to be broken down.

Moreover, as the CDC explains, the agent is filterable. Bastian notes that Spiroplasma can go through a 50-nm filter; by comparison, herpesvirus is about 100 nm in size. This organism's survivability fits the transmissibility model for TSE, says Bastian. In addition, he points out that Spiroplasma replicates in mammalian tissue. Some critics of the autoimmune-disease and Spiroplasma theories, however, state that established data and scientific proof to support them are lacking, and that PrP, as a self-protein, is never recognized by the immune system as a homologous host.

A Disease Vector?

Could a vector for scrapie and, by extension, TSE and vCJD, be lurking in grass and hay that sheep eat? Sometimes, scrapie-free sheep placed in contact with infected sheep develop the disease. Also, scrapie can occur in a previously scrapie-free flock, and scrapie-free sheep put on farms where there had been infected sheep--even though no sheep were there for up to three years--have developed scrapie. Does this indicate the presence of a reservoir for an infectious agent in the environment?

Scrapie occurs frequently among sheep in Iceland. In a letter printed in the 20 April 1996 issue of Lancet , Henryk Wisniewski, director of the New York State Institute for Basic Research in Developmental Disabilities and Mental Retardation in Staten Island, New York, and his colleagues there and at the University of Iceland in Reykjavik described a study of grass and hay from farms at which sheep had had scrapie. First, they removed all such sheep and confirmed scrapie by analysis for PrP Sc . Then they collected several species of hay mites from up to five scrapie-affected Icelandic farms and froze them at -20 o C. Mite samples were thawed, decontaminated, washed with phosphate-buffered saline (PBS), and centrifuged. Pellets were then ground and suspended in PBS. Centrifuged supernatant was collected for intracerebral injection into female Compton white mice aged 4-6 weeks. The pellet suspension and centrifugation procedure was repeated and the supernatant was then injected into mice intracerebrally or intraperitoneally.

Mites isolated from these Icelandic farms were shown to contain prion infectivity, as assayed by inoculation into mice. These prions appear to possess characteristics that distinguish them from other known strains of scrapie agent, such as long incubation times, unusual clinical manifestations of disease, and gel profiles of protease-resistant prions.

In their letter to Lancet , Wisniewski and his colleagues suggest the possibility that this evidence supports the concept of an invertebrate reservoir or vector for some strains of prions. He points out that extensive precautions to avoid contamination were taken and that the methods he and his colleagues used suggest that partially purified mite preparations contained antigenic sites similar to those in PrP. Wisniewski also suggests that hay mites acting as a vector or reservoir might have played a role in the continuing occurrence of BSE even after British authorities forbade the use of ruminant animal products for cattle feed. Bastian has suggested that Spiroplasma in the hay mites are the likely cause of this experimental infection.

Testing for the Agent

Researchers in government, academia, and industry are seeking methods of testing for PrP Sc or whatever the disease agent may be. For example, Mary Jo Schmerr, a research chemist at the National Animal Disease Center in Ames, Iowa, and her colleagues are investigating the use of organic solvent extraction together with high-performance liquid chromatography to extract sheep tissues, including brain, spleen, lymph nodes, and tonsils. She told a post-TSE conference symposium on testing that capillary electrophoresis will be used to characterize proteins in the extracts and test for prion protein. In addition, an immunoassay with fluorescent-labeled peptides from prion protein with laser-induced fluorescence detection will be conducted to detect the presence of prion protein.

pills Pill scare . An EU ban on orally ingestible medicines containing animal products could lead to a worldwide shortage of such drugs.

Also at the symposium, Lily Yang, chairman and chief executive officer of Neuromark Corporation in Los Altos, California, who also was chairperson of the TSE conference, described a rapid ante-mortem immunoassay, 14,3,3 marker in cerebrospinal fluid, that has a 99% sensitivity and 98% specificity in CJD patients with confirmed brain pathology. She added that a similar study of BSE-afflicted cattle from Great Britain showed a similar high correlation. Yang explained that the antigen is shared by other animals affected by TSE, including sheep and monkeys.

In December 1997, Prusiner, along with Fred Cohen and Stephen DeArmond, also of the University of California at San Francisco, announced the development of a strain of genetically altered mouse that responds within 250 days to TSE infection and reliably develops the disease. Most importantly, the mouse will respond to lower doses of infection.

"This model," says Cohen, a professor of cellular and molecular pharmacology, "is sensitive enough that it is now possible to make an accurate determination of the amount of BSE prion in brain and other tissues--muscle, pancreas, liver, and intestine--that are commonly consumed by humans." This is a particularly critical achievement because nonbrain tissues, such as muscle, contain lower amounts of prions. The model will also make possible more accurate scrutiny of drugs and other medicinal products derived from cattle, such as collagen for plastic and reconstructive surgery, and gelatin for food and the production of drug capsules.

Having such a sensitive test also opens up the door for conducting epidemiologic studies on the frequency of BSEs in countries such as the United States and Canada that are thought to have been spared, says DeArmond, a professor of pathology and neurology. And findings in this regard will determine whether it will be important to produce transgenic cattle that are resistant to prions.

Cold Comfort for North Americans

North Americans may derive some cold comfort from indications that, to date, BSE and vCJD have not appeared in North America. So are North Americans safe from TSE? One should not say yes unequivocally. For example, some wild ruminants in the western United States and Canada are vulnerable to chronic wasting disease, a scrapie-like affliction.

In addition, SE has been reported in a variety of large and small mammals. While conducting a study of CJD in south Florida, Joseph Berger, chairman of the department of neurology at the University of Kentucky in Lexington, observed an affected patient who was originally a native of Kentucky and had a history of eating squirrel brains. Subsequently, five other patients with confirmed or suspected CJD with a history of squirrel brain consumption were identified in rural Kentucky, in collaboration with Eric Waisman. These case reports, detailed in a letter published in the 30 August 1997 issue of Lancet , have suggested the possibility of transmission of CJD by consumption of brains of wild animals. Berger cautions that this observation will require confirmation by studies of larger populations, and by a search for a scrapie agent in the brains of squirrels, which so far have not been reported as having spongiform encephalopathies. In the meantime, he counsels caution in the ingestion of the brains of this arboreal rodent.

Did mad cow disease become a foodborne human health threat? Absent the irrefutable identification of a TSE-causing agent, it's probably prudent to assume that this has occurred. Perhaps Berger's admonition should be applied to brains and certain other tissues of all animals: all unnecessary nonfood as well as food contact should be rigorously avoided, and TSE-suspect tissue should be disposed of according to the strictest protocols that apply to highly infectious waste.

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Editor: Susan K. Boyer, RN
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