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Prions: a brief overview.

A note from the authors: Prions, "Mad Cow Disease" and Creutzfeldt-Jakob disease are terms most of us in the clinical laboratory are familiar with, but we don't think about them much, if at all, until someone calls the laboratory or pathology about a patient with suspected CJD! What do we do and why? We wrote this review as an update of the unique groups of "agents" and to remind us all of the "why."


Mad Cow Disease became a household phrase and world-wide news event in 1986 when farmers in England recognized that something untoward was happening to their cattle. As the story unfolded, it became clear that the root of the problem could be traced back to the feeding practices: infected brains, nervous systems, and blood of infected cattle were being fed to other cows. It has been estimated that nearly one half million infected or at-risk cattle were on the market during the 1980s.

The human part of the puzzle became known as variant Creutzfeldt-Jacob Disease (vCJD). People who became infected with vCJD had either eaten contaminated beef or perhaps inhaled the disease while using fertilizer made from infected cattle tissue. By 2004, nearly 160 people had developed vCJD and died; however, whether all of the infections have manifested themselves is unclear. England and perhaps the world may face a large number of cases in the next 20 to 30 years.

In the U.S., only a handful of cases of Mad Cow Disease-or bovine spongiform encephalopathy (BSE)-have occurred. The United States government has taken extreme precautions to prevent any cases of vCJD in North America by banning the use of downer cattle for any type of food and by testing and slaughtering many suspected animals. An article in the Atlantic magazine reported that the U.S. tested 20,526 cows and killed 35 million just to keep vCJD from spreading in North America.

Transmissible Spongiform Encephalopathies (TSE) are chronic degenerative neurological diseases characterized by the accumulation in the brain of an abnormal form of a cellular glycoprotein known as PrP or prion protein (proteinaceous infectious particle). This abnormal form is usually designated PrP TSE or [PrP.sup.Sc] and differs from normal PrP ([PrP.sup.C]) in its shape.

There are a number of different spongiform encephalopathies in humans, including: several forms of Creutzfeldt - Jakob Disease (CJD), Kuru, Gerstmann - Straussler-Scheinker Syndrome (GSS), and Fatal Familial Insomnia (FFI). TSE diseases in animals include: bovine spongiform encephalopathy (BSE) in cattle; Scrapie in sheep and goats; chronic wasting disease (CWD) in deer and elk; transmissible mink encephalopathy (TME) in farmed mink; feline spongiform encephalopathy (FSE) in cats; and spongiform encephalopathy of exotic hoofed animals in zoos.

The first reported TSE was Scrapie: a neurological disease of sheep first described in England, France and Germany in the early 18th century. Affected sheep rubbed against trees or posts, thus the name "Scrapie." But it was an epidemic disease described in 1957 that piqued the interests of many of the world's microbiologists and infectious disease specialists.

This epidemic was among the Fore tribe in Papua New Guinea, whose rituals involved eating the body-including the brain-of recently deceased members of the tribe. The neurological disease in this tribe was called Kuru. Hadlow noted the similarity of pathology of brain tissue in Kuru to that of Scrapie. Subsequent work in an animal model raised the possibility that a disease like Scrapie could be transmitted; could exist in humans; and that it may involve eating one's own species.

The most widely accepted theory at the time involved an unknown "slow" virus. Research into this phenomenon was limited for a number of years, but in 1982 Stanley B. Prusiner's highly controversial research in "prions" began to make international news.


Genetics and Molecular Properties of Prions

The genetic focus of prion disease lies in the Prnp gene, which is located on chromosome 20 in humans and is a member of the Prn gene family. The Prn gene family also includes the Sprn gene and Prnd gene. These genes encode respectively for the Shadoo and Doppel proteins. The Prnp gene itself consists of two exons and an open reading frame that encodes for the 253-amino-acid-long prion protein known as PrP.

The functional protein, [PrP.sup.C], is post-translationally formed by the cleavage of a 22 amino acid N-terminal peptide and a 23 amino acid C-terminal peptide, leaving a protein of 209 amino acids in length. Mature [PrP.sup.C] proteins are bound to cell membranes by the addition of a glycosyl-phosphatidyl inositol (GPI) anchor that occurs during the cleaving of the C-terminus.

The mature form of the protein is composed of two domains, the N-terminal domain and C-terminal domain. The N-terminal domain is composed of five octapeptide repeats that are rich in proline and glycine, the first of which is termed a "pseudorepeat" because it lacks histidine. The remaining four repeats are all identical sequences. It has been shown that the N-terminal domain holds the ability to bind copper ions-along with other polyan-ions such as hemin-by forming [beta]-turn conformations composed of glycine, histidine and tryptophan residues. This region appears to be an evolutionarily conserved motif across species. NMR studies suggest that this region is highly flexible and disordered due to its lack of secondary structures.

The C-terminal domain is comprised of three [alpha]-helices and two short [beta]-strands that form an antiparallel [beta]-sheet. The bulk of this domain is composed of the latter-two helices (termed 2 and 3) that are covalently held together by a disulfide bond. Also present are two variably utilized glycosylation sites and hydrophobic regions that allow the mature [PrP.sup.C] protein to obtain a small and unusual hydrophobic core. Overall, the C-terminal domain appears to be of great importance in terms of disease susceptibility and protein interactions that may lead to a disease state.

PrP is found in many adult tissues, but is most highly concentrated within the immune and central nervous systems. Despite this, its function is still largely unknown. Experimental mice lacking the Prnp gene have been shown to function normally and show no gross phenotypic abnormalities. Despite this finding it has been suggested that PrP may protect cells against oxidative stress and function as part of a modulation pathway important to cell survival. More recent studies have focused on the potential role of PrP in the long-term maintenance of neuronal function.

The abnormal, misfolded or infectious form of PrP is known as [PrP.sup.Sc]. The actual structure of [PrP.sup.Sc] is still poorly understood, but unlike monomeric [PrP.sup.C], [PrP.sup.Sc] is thought to be oligomeric in nature. [PrP.sup.Sc] contains less [alpha]-helical content and more [beta]-sheet structure than [PrP.sup.C]. This property makes [PrP.sup.Sc] highly resistant to proteolytic enzymes such as proteinase K; and allows it to form highly insoluble aggregates of homologous proteins, known as amyloid plaques, in tissues. These deposits are thought to be destructive to tissues and lead to prion-associated diseases.

The events responsible for the conformational conversion of cellular [PrP.sup.C] to the misfolded form [PrP.sup.Sc] are currently under investigation; however, two protein-based replication processes have been proposed. The first proposed process is known as the template assisted or heterodimer refolding model. Under this model [PrP.sup.Sc] exists as a more thermodynamically stable monomer than [PrP.sup.C]. Thus, when [PrP.sup.Sc] is spontaneously created or exogenously introduced it acts as a monomeric-template, forming a heterodimer with cellular [PrP.sup.C]. That [PrP.sup.C] is then refolded into a more thermodynamically stable [PrP.sup.Sc] monomer, and the process can then repeat itself. This model is possible, but highly unlikely because the infectious entity of [PrP.sup.Sc] is known to be oligomeric in nature and no evidence exists for a stable [PrP.sup.Sc] monomer.

The more widely accepted model or process is known as the nucleated polymerization mechanism. In this model the transformation of [PrP.sup.C] to [PrP.sup.Sc] is reversible, but [PrP.sup.Sc] is thought to be much less stable. Here it is proposed that through a thermodynamically unfavorable process, an oligomeric nucleus is formed when a protein adapts a [PrP.sup.Sc]-like structure capable of recruiting pools of monomeric [PrP.sup.C] molecules to it. As the [PrP.sup.C] molecules are added they are folded into the [PrP.sup.Sc] conformation and polymerization occurs, making the process more favorable to continue. As the [PrP.sup.Sc] aggregates grow they begin to fragment into smaller units that in return recruit more pools of [PrP.sup.C], and the process continues to repeat itself.

Prion Diseases in Humans

Creutzfeldt-Jakob Disease

Creutzfeldt-Jakob Disease (CJD) is the most common human prion disease. It can arise sporadically or as a result of a genetic mutation, and can also be transmitted. Most patients suffering from CJD present with confusion and memory loss that eventually leads to severe cortical dementia. These symptoms are usually found in combination with variable ataxia and myoclonus; however, the clinical presentation varies tremendously between individual cases due to the variety of etiologies and disease phenotypes or strains. This is most evident in families with the same pathogenic PRNP mutation. Other common symptoms may include: weakness, rigidity, bradykinesia, tremor, chorea, alien hand syndrome, and sensory disturbances. Complaints of fatigue, headache, sleep disturbance, vertigo and behavioral changes are less common.

Vacuolation or spongiform degeneration in the grey matter of the six cortical layers of the cerebral cortex or within the molecular layer of the cerebellar cortex is the hallmark pathological feature of CJD. These spongiform vacuoles generally range in size from 5 to 25mm. The brain tissue of infected individuals will often show a reactive gliosis in the absence of an inflammatory response, and protease-resistant PrP deposits are often easily detected.

Sporadic CJD

Sporadic CJD (sCJD) was first described in 1920 and accounts for those cases of CJD that have neither detectable familial nor infectious causes. sCJD is responsible for nearly 80% of the total cases of CJD per year, occurring at a rate of approximately 0.5 to 1 per million population per year. Although it generally affects individuals in their seventh decade of life, it has been seen in patients ranging in age from 17 to 90 years old.

The disease course of sCJD is rapidly progressive and lasts on average 4 to 6 months. All patients suffering from sCJD are affected by a rapidly progressive dementia followed by a host of other neurological symptoms. By the end most patients become completely unresponsive to outside stimuli. The leading hypotheses on the cause of sCJD are the spontaneous conversion of [PrP.sup.C] to [PrP.sup.Sc] during a rare stoichastic event in an otherwise healthy human, or from a somatic mutation in the Prnp gene of a single neuron. Although it has been transmitted to laboratory animals, no evidence of horizontal transmission has been established in humans.

Familial or Genetic CJD

Inherited or Familial CJD (fCJD) was first described in 1924 and accounts for 15% of the total reported cases of CJD. However, approximately 50% of the cases reported do not show any family history of disease, leaving some to use the term "genetic CJD" more frequently. Today nearly 40 autosomal dominantly inherited pathogenic mutations of the Prnp gene have been documented to cause fCJD. The most common of these mutations is found in Jews of Libyan and Tunisian origin, who have an incidence of CJD about 100 times higher than the worldwide average.

fCJD is caused by the inherited E200K point mutation, where the amino acid glutamic acid is replaced by lysine at position 200. Unlike sCJD, fCJD appears to manifest itself at a younger age; however, a variable and often lateage onset of the disease may be overlooked. Patients suffering from fCJD typically have a more protracted course of a diseased state, lasting from 1 to 5 years.

Iatrogenic CJD

Iatrogenic CJD (iCJD) is defined as cases of CJD spread human-to-human through contaminated medical procedures. It was first described in 1974 in a corneal transplant patient. By the end of the 1990s there were over 200 cases of iCJD, most of which were attributed to two major epidemics.

The first epidemic of iCJD began in the 1950s and was associated with the therapeutic use of human growth hormone obtained from pools of cadaveric pituitary glands. It affected a total of 206 young, growing individuals-28 of whom resided in the United States, and the majority within France. The mean incubation period of affected individuals was 15 years, with a range of 4-36 years. Overall the epidemic seems to have come to an end, and in 1985 the use of cadaveric human growth hormone was replaced by growth hormone produced using recombinant DNA technology.

The second epidemic of iCJD was attributed to the use of heterologous cadaveric dura mater in neurosurgeries between 1979 and 1996. It totaled 196 cases, 63% of which came out of Japan-who finally banned the procedure in 1997 in favor of better alternatives, including synthetic dura matter. The mean incubation period of affected patients was 11 years, with a range of 16 months to 23 years. Today, the threat for new iCJD cases appears to be minimal due to current knowledge and present awareness; however, a peak in incidence may yet occur due to iCJD cases with long incubation periods.

Variant CJD

In 1995 the first cases of variant CJD (vCJD) began to surface in the United Kingdom. Epidemiologic, pathologic and biochemical studies have strongly linked this form of CJD to the human consumption of BSE-contaminated cattle products. Most notably, the fragment size and glycoform ratio of the [PrP.sup.Sc] is very similar to that in BSE-infected cattle.

Between 1980 and 1996 an estimated 750,000 BSE-infected cattle were thought to have been slaughtered. To date, a total of about 219 cases of cross-species transmission have been documented in eight European countries and four non-European countries including the United States, Canada, Saudi Arabia and Japan. However, 172 of these cases originated from the United Kingdom, where the epidemic peaked between 2000 and 2003. To date, it appears that all but one of the reported primary cases of human vCJD appear to be limited to subjects homozygous for the amino acid methionine at codon 129 of the Prnp gene. This polymorphism is known as the 129MM genotype, and these findings now strongly suggest that individuals harboring this genotype have an increased genetic susceptibility towards vCJD.

Overall, vCJD differs from most other forms of CJD in that it generally affects patients ranging in age from 16-39 years, with a mean age of onset at 29 years. The progression of the disease usually lasts about 18 months on average. Its symptoms differ in that notable psychiatric features, such as apathy and depression, arise alongside the presence of painful distal sensations. Histological examinations show the presence of dense core PrP plaques surrounded by spongiform change, known as "florid plaques," which are a unique pathology of vCJD.

Today, a declining incidence in vCJD cases can most likely be attributed to public health measures taken to prevent contaminated food products from entering the human food chain. However, it should be noted that secondary infection, or human-to-human transmission, of vCJD through the transfusion of blood and blood products has been documented. This has further prompted concerns for both the presence of an asymptomatic carrier state for vCJD and the safety of the blood supply.

Gerstmann-Straussler-Scheinker Syndrome

First described in 1936, Gerstmann-Straussler-Scheinkerr (GSS) syndrome is a familial human prion disease caused by the archetypal mutation P102L of the Prnp gene. This pathogenic mutation results in the substitution of thymine for cytosine at codon 102, resulting in the coding of a leucine amino acid instead of the normal proline. However, today up to nine other point mutations have been associated with GSS. It has an incidence of 1 in 100 million of population per year and generally affects persons of 30-60 years of age. The disease progression lasts anywhere from 2-7 years, and suffering is most notably from ataxia. Cognitive decline, leg weakness, and lower limb pains are often less prominent. Additionally, psychiatric symptoms, pyramidal and extrapyramidal signs, and late stage dementia are seen, depending on the disease phenotype present. The most common pathological features of GSS include numerous amyloid plaques, spongiform changes, neuronal loss, astrocytic microgliosis, and neurofibrillary tangles.

Familial Fatal Insomnia and Sporadic Fatal Insomnia

Familial fatal insomnia (FFI) is a rare human prion disease caused by the D178N point mutation of the Prnp polymorphism M129V. The mutation results in a change of the normal amino acid aspartic acid to asparagine at codon 178, but must be coupled with the same allele as the methionine codon at position 129. If codon 178 encodes for a valine, then the mutation will result in fCJD. The disease was first described in Italy, but cases have now been described in Germany, Austria, Spain, United Kingdom, France, Finland, United States, Australia, Japan, China and Morocco. Equally occurring in both men and women, about 100 FFI cases have been described in 40 families to date. The average age of onset is 49 years old, with a range of 20-49 years old. Its course can last anywhere from 7-33 months, but on average is about 1-2 years.

The cardinal symptoms of FFI include insomnia and lack of attentiveness. Excessive daytime sleep often occurs due to lack of nocturnal somnolence. Hypertension, excessive sweating, evening fevers, salivation and impotence may accompany the major symptoms. Patients later suffer from vivid hallucinations and dreaming. Other symptoms that have been noted include myoclonus, pyramidal dysfunction, and ataxia. Although it affects both the 129MM and 129MV genotypes equally, the array of symptoms present during the course of FFI differs between the two. The hallmark pathological features of FFI include focal thalamic and olivary gliosis with neuronal dropout.

In 1999 a form of sporadic fatal insomnia (sFI) was described. sFI appears to be much like FFI in that the patients suffering from the disease show similar clinical presentation, namely insomnia. However, sFI patients have all lacked family history and the D178N mutation entirely. To date, only 24 sporadic forms of FI have been documented in various countries. All patients have been 129MM homozygous.


Kuru is a prion disease of humans that is of particular interest because of its historical place in our understanding of this unique constellation of diseases. This fatal neurologic-degenerative disease was only found among a unique linguistic group or tribe known as the Fore people. Although focused in the Fore, there was some disease (approximately 20% of all cases) reported in their neighboring people in the Okapa region of the Eastern Highlands Province of Papua New Guinea.

The story of this unexpected disease began when Australian administrators began surveying the island in the early 1950s. Early reports by anthropologists visiting the Okapa region in 1952 and 1953 indicated the existence of an unusual debilitating disease among the people. An early description of Kuru was made in a report in 1954 by W.T. Brown in the Kainantu Patrol Report No. 8 covering January to February 1954.

Research began in earnest in 1957 with the mapping of the geographic distribution of the disease by Dr. Michael Alpers and anthropologist Shirley Lindenbaum. Their historical research with the Fore suggested that Kuru may have originated around 1900 from a single individual who lived on the edge of the Fore territory. It was theorized that this individual had spontaneously developed some form of Creutzfeldt-Jakob or other prion disease. Alpers and Lindenbaum demonstrated that Kuru spread rapidly in the Fore people due to their funeral practices: in this group of inter-related people, relatives consumed the bodies of the deceased to return the "life force" of the deceased to the people.

Dr. Vincent Zigas, District Medical Officer for Papua New Guinea, began work in the region in 1957 and was soon joined by Dr. Daniel Carleton Gajdusek of the United States National Institute of Health. In 1964 Dr. Alpers collected post-mortem brain tissue samples from an 11-year-old Fore child who had died of Kuru. Working with Dr. Gajdusek, they injected two chimpanzees with the infected material. Within two years, one of the chimpanzees developed Kuru. This demonstrated that the unknown "factor" was transmitted through tissue and that it was capable of crossing the species barrier. In 1976 Gajdusek, along with Baruch S. Blumberg, was awarded the Nobel Prize in Medicine for showing that Kuru was transmissible.

With the elimination of the death ritual through Australian law enforcement, Alpers showed that Kuru was declining among the Fore by the mid-1960s. Interestingly, cases continued to appear for several decades more, and the last sufferer died in 2005. The mean incubation period of the disease is 14 years, and cases were reported with latencies of 40 years or more for those who were most genetically resilient.

A genetic and clinical assessment led by Simon Mead of University College London concluded that the survivors of the epidemic in Papua New Guinea carried a prion-resistant factor. Mead's group has shown the source of immunity to be the inheritance of a genetic variant of prion protein G127V.

Prion Diseases in Animals


First documented in 1732, Scrapie was the first of the transmissible spongiform encephalopathies described in animals. It is a fatal neurodegenerative disease of the CNS in sheep, goats and mouflons (wild sheep). The name "Scrapie" comes from the classic itching symptom that infected sheep display, which results in broken or pulled wool from compulsive rubbing and scraping of their wool against rocks, trees and fences. Today Scrapie is found worldwide and no one breed is known to be immune. However, the most commonly affected breed is the Suffolk, followed by other black-faced meat breeds.

Originally found in Great Britain and Western Europe, Scrapie has now spread to flocks of sheep worldwide. Currently, only Australia and New Zealand are recognized by the USDA as being "Scrapie free." In 1947 the United States reported the first cases of Scrapie in Michigan after sheep arrived through Canada from Great Britain. Today the disease is estimated to cost the U.S. sheep industry over $20 million a year and has affected approximately 1,600 flocks in the United States. Seven cases of Scrapie in goats have also been reported in the United States.

Much about the transmission of Scrapie is still poorly understood, but it is only one of two TSEs known to effectively spread within a species under both natural and near-natural conditions. Thus, it highlights the ability of prion diseases to behave as infectious entities. Transmission is known to be both horizontal, through direct contact; and vertical, from the mother to her offspring. Infectious [PrP.sup.Sc] particles can be found in the secretions and excretions of infected sheep. However, infection is most common in the first six months of life when the lambs come into contact with the birthing fluids and placenta of the ewe.

Like all TSEs, the clinical symptoms of Scrapie appear to be largely influenced by the strain present and the genetic background of the individual affected. Symptoms are the result of damage to nerve cells that leads to neuronal dysfunction. The early signs of Scrapie typically occur around 2 to 5 years after exposure and include: anxiety, tremors of the head and neck, apprehension, weight loss, loss of coordination, and other behavioral changes such as a lack of desire to flock. This is followed by a number of late symptoms including compulsive itching and scratching, high stepping, stumbling and falling, and teeth grinding. Death is ultimately inevitable and almost always occurs within 6 months of clinical onset.

The diagnosis of Scrapie is generally made by observation of the clinical symptoms and confirmed by the pathological examination of affected brain tissues taken after death. The necropsy of infected brain tissues shows spongiform vacuolation or holes in the brain, and the accumulation of abnormal proteins resulting in amyloid plaques. A newer diagnostic method that is becoming important in the control efforts of Scrapie is the eyelid tissue test. Here lymphoid tissue from the third eyelid of live animals of 12-14 months of age is taken in the hopes of detecting infected individuals at a young age and isolating them from the rest of the flock.

To control Scrapie, the best method is to keep a closed-ewe flock by limiting exposure occurring through shared environments such as birthing facilities. Since the disease appears to show genetic susceptibility, the use of genetic selection techniques have also proven to be effective. These include using genotype testing to produce resistance in flocks with susceptible ewes with rams of the resistant genotypes. The improvement of hygiene in lambing facilities is also important as a control method. This is accomplished by providing individual birthing pens, disinfection, and quick and proper disposal of the placenta and other fluids produced during delivery.

Bovine Spongiform Encephalopathy

BSE is most commonly known as "Mad Cow Disease" to the general public. It is a fatal neurodegenerative disease found in cattle, characterized by spongiform vacuolation in the brain due to the accumulation of abnormal proteins. Cattle infected with BSE generally show gait abnormalities, particularly of the hindlimb (ataxia), along with aggressive behavior, apprehension, tremors and hyperreactivity to stimuli.

The first outbreak of BSE began in the United Kingdom in the 1980s and peaked in 1992 with nearly 1,000 cases a week in the UK. Nearly 185,000 cases of BSE have been confirmed in the United Kingdom alone. The disease is now known to occur worldwide, but with a much smaller prevalence outside the UK. For example, in North America only 19 cases had been confirmed up until 2008. However, the disease has once again gained recent headlines for two atypical cases emerging in California.

The main route of transmission during the height of the BSE outbreak was through the practice of using cattle feed containing meat and bone meal contaminated with infectious prions. Meat and bone meal was thought to improve the amino acid profile of animal feed and thus result in better growth. An immediate ban on this practice soon resulted in a progressive decline of the epidemic. So far there has been no evidence for horizontal transmission of BSE in cattle, and prions have not been found in the milk, semen, or embryos of infected cattle.

BSE has affected nearly 0.2 million Holstein-Freisian cattle worldwide to date. BSE is generally found at 4 to 5 years of age, and symptoms can begin as soon as 2 years after exposure. However, the incubation period has been shown to be up to 8 years.

Studies have shown that BSE transmission has naturally occurred in sheep and goats due to either the consumption of contaminated feed containing cattle-derived protein supplements or from direct contact with infected individuals. Most shocking is the ability that BSE has historically shown to contaminate the human food chain. Humans exposed to contaminated beef products can acquire a human form of BSE, known as vCJD due to its similarities with other human prion diseases. These contaminated products often contain the brain, spinal cord, or digestive tract of infected animals. Cats have also ultimately fallen victim to the BSE epidemic with cases of FSE peaking in the 1990s.

Feline Spongiform Encephalopathy

Feline Spongiform Encephalopathy (FSE) is a TSE that occurs in both domesticated cats and other wild felines of the family Felidae. Since the 1990s nearly 100 domestic cats and 29 captive wild cats have fallen ill with FSE, most of whom have originated from the United Kingdom. Other European countries with reported cases of FSE include: Ireland, Norway, Liechtenstein, Switzerland, France, Australia and Germany.

All cats with reported FSE have been greater than 2 years of age and have demonstrated a variety of clinical manifestations. Most cats initially demonstrate behavior changes, depression, restlessness and neglect in coat grooming. Major behavioral changes include fear, timidity, hiding and uncharacteristic aggressiveness. These behavioral changes are soon followed by ataxia and abnormal gait, mainly of the hind limbs. Affected individuals also have increased sensitivity to sound and touch. Other symptoms may include excessive salivation, tremors and convulsions.

Death in domestic cats occurs within 3-8 weeks of clinical onset, and in larger cats within 8-10 weeks. The generalized histopathology reveals spongiform degeneration in the neuropil of the brain and spinal cord. Deposits of PrP and florid plaques are detectable with immunohistochemistry in numerous areas including the central, peripheral, and lymphoreticular systems; kidneys; adrenal glands; and retina.

It is hypothesized that FSE arose in cats after exposure to feed containing BSE-infected cattle carcasses, namely the bovine spleen and CNS tissue. This can be supported by the fact that all but one of the FSE cases to date occurred in cats exposed to contaminated feed prior to the ban put in place in 1990. Also, mice inoculated with brain homogenates from cats with FSE showed neuropathological lesions and incubation periods similar to those in BSE-infected cattle. More importantly, strain typing further supports the hypothesis that BSE-contaminated foods affected more than one species.

Transmissible Mink Encephalopathy

Transmissible Mink Encephalopathy (TME) is a rare TSE found in farm-raised mink, a small semi-aquatic animal valued in many countries for its fur. The first cases of TME arose in Wisconsin and Minnesota in 1947. Other outbreaks of TME occurred in the 1960s and 1970s, but the most recent was in 1985 where nearly 4,300 of the 7,300 adult mink on a farm located in Stetsonville, Wisconsin were affected over a 5-month period. To date, TME has been found in the USA, Canada, Finland, Germany and the former USSR.

Although to date the origin and etiology still remain unclear, TME is presumed to be an orally acquired TSE. Today the most plausible explanations for outbreaks of TME have been through the use of contaminated feeds containing either Scrapie-infected sheep carcasses or sickened downer cattle. Once infected, minks housed together may acquire the infection through either cannibalism or biting. No environmental modes of exposure have been shown to help spread TME amongst farms. Vertical transmission does not seem to play a role since TME has only been detected in adult mink.

TME is almost always associated with a high mortality rate, sometimes upwards of 100% during outbreaks. Clinical manifestations have been well documented. With an incubation period of about 6 to 12 months, the early symptoms of the disease course typically begin with behavioral changes such as increased aggressiveness, hyperesthesia, depression and restlessness. Lack of parental care and coat grooming, along with the uncleanliness of cages, can be seen as affected minks will often soil their nests and scatter feces throughout the cage. Some mink may even display difficulty eating and swallowing during the early stages of the disease.

The early stages are soon followed by more troublesome symptoms including: ataxia, abnormal gait, loss of coordination, tremors, jaw clenching, curved tail posture, and compulsive behaviors such as the mutilation of specific objects or themselves. During the late stages of the disease, neurological symptoms begin to become prominent and mink may convulse at times. Often times they become unresponsive and somnolent: pressing their heads against the cages for hours. Death ultimately occurs within 2 to 8 weeks of the onset of symptoms. Neuropathological features of TME include spongiform degeneration in the neuropil of the brain, astrocytosis and the formation of PrP deposits of the TME agent.

Chronic Wasting Disease

Chronic Wasting Disease (CWD) is a TSE found in members of the family Cervidae. First described in 1967 in captive mule deer of Colorado, it wasn't until the 1980s that the disease was defined as a spongiform encephalopathy. Most notably, this specific TSE was found to be unique in that it affected both farmed and wild animals. CWD has to date been known to impact mule deer, whitetailed deer, black-tailed deer, Rocky Mountain elk, and less frequently moose populations.

Originally considered to be just a rare and exotic disease, today CWD has gained much attention as it continues to expand both its prevalence rates and geographic distribution due to its highly contagious behavior. Although the origins of CWD are not well understood, it is hypothesized that it arose after either spontaneous mutation of normal cervid PrP into [PrP.sup.Sc], or through a modification of Scrapie strains that allowed the introduction of the disease into cervids. Strikingly, more than one of these [PrP.sup.cwd] strains may exist amongst an affected herd.

In the United States alone CWD can be found in 17 states and is considered endemic within Colorado and Wyoming. Yet, it has been described as far south as New Mexico and as far east as New York and West Virginia. CWD has also been described in Canada, with cases occurring in both Alberta and Saskatchewan. Outside of North America the only other reports of CWD have come from South Korea, where it was described on a farm containing imported animals from Canada. Its absence in native European roe deer, a species known to be susceptible via experimental transmission, may be misleading due to limited surveillance data being captured at this time.

Horizontal transmissibility and the ability for infectious prions to persist in the environment appears to be the most pronounced characteristics of CWD, making control of the disease an extremely complicated situation. Both the indirect and direct transmission modes have been shown to be effective. CWD-causing prions can be shed via decomposing carcasses, feces, urine and saliva. They are also found throughout the diseased host including the skeletal muscle, blood and a wide range of other tissues in CWD-infected cervids, some of which appear to be asymptomatic.

More importantly, it is their stability within the environment that allows the prions to persist. They are not only resistant to destruction by conventional methods such as chemical disinfectants and proteases, but also to natural conditions including UV irradiation, freeze-thaw cycles, as well as extracellular enzymes produced by bacteria and fungi. This allows them to potentially threaten the pastures and water sources that herds frequent for long periods of time.

CWD appears uniformly between sexes of captive deer herds, but in the wild mule deer populations the prevalence appears to be higher amongst mature males. Prevalence in wild herds can grow as high as 50%, and more astoundingly up to 90% in affected captive herds. Affected animals are generally older than 2 years of age, with an average of 3-5 years old. The incubation period of CWD has a range of 16 months to 5 years, with death usually occurring within one year of the onset of symptoms. These symptoms can include behavioral changes, weight loss, excessive pacing, excessive salivation, altered stance, and hyperexcitability. The histological pathology found in animals affected by CWD is consistent with that of other animal prion diseases.

The zoonotic potential for CWD transmission to humans is a reasonable concern considering the popularity of deer hunting and the consumption of venison in the United States. This is especially true given the highly contagious nature of the prion in cervids and the increasing prevalence of CWD. Lab studies have shown that experimental intraspecies transmission is possible to non-cervids; however, to date no known natural transmission has occurred outside of the known hosts. Thus, the zoonotic potential is still considered a low probability at this time.

Miscellaneous Prions


Several prion proteins have been identified in fungi in the last 10 years, most in Saccharomyces cerevisiae. These prions have provided a convenient model to study prions in man and other mammals. Fungal prions are naturally occurring proteins that can undergo a structural conversion that becomes self-propagating and transmissible or infectious. Some of these proteins do not appear to be associated with disease or pathology in the host, but rather may possibly have a beneficial or evolutionary role.

Synthetic Prions

In the journal Science in 2010, Wang and associates, building on previous publications, reported using recombinant mouse PrP purified from Escherichia coli to create a synthetic or recombinant prion with the characteristics of pathogenic prions, i.e., aggregate-forming, protease-resistant and self-perpetuating. After intracerebral injection of this prion, a significant number of wild-type mice developed neurological signs and showed the classic neuropathology of prion disease. These experiments support the fact that the infectivity in mammalian prion disease results from an altered PrP.


So far, only beef cows and sheep have been identified as significant potential sources of prion infections from food consumption. Other major food sources such as fish and poultry appear to be free of prion contamination. However, studies are limited. Sequence data documenting the existence of PrP's in fish has revealed the presence of PrP-like proteins in various tissues of fish species such as gilthead sea bream (Sparus aurata), Japanese sea bass (Lateolabrax japonicus), Japanese flounder (Paralichthys olivaceus), and Fugu (Takifugu rubripes). PrP's can be found in the adult fish brain, muscle, skin, heart and gills. PrP sequences among bony fish are relatively well conserved across groups (50-60% similarity), but are only 20-25% similar to mammalian proteins. It was this significant difference from mammalian proteins that complicated the search for prion proteins in fish. The first PrP-like proteins were identified in Takifugu rubripes using genomic database analysis. At least two distinct proteins, PrP-1 and PrP-2, have been identified in fish. Additionally, proteins that may have similar functions have been reported in other fish.

Early studies by Loredana showed that fish tissues taken at different time points after oral or parenteral inoculation with PrP's cannot provoke Scrapie disease after intracerebral inoculation in recipient mice. In a more recent study, Dala Valle et al., investigating prion infectivity in fish, found that PrP's can be absorbed by the intestinal mucosa and can persist in the fish gastrointestinal tract for up to 3 days in pyloric caeca and for up to 7 days in the distal intestine. In these studies the PrP did not remain longer than 15 days in the fish intestine and did not appear to cross the intestinal barrier. In addition, studies by Salta and associates using sea bream demonstrated differing results. A number of fish that had been force-fed Scrapie-infected brain homogenates developed histological signs of abnormal protein aggregates in the brain after 24 months. In fish force fed BSE-infected tissue, abnormal deposits were seen at 9 months and progressed over time. By 24 months, three of five fish had significant amyloid-like structures in the brain compared to controls. Interestingly, there were no observable "symptoms" with the experimental group of fish.

Further work is needed to define the risk, but clearly there is cause for concern about using cow or sheep derived proteins (and perhaps fish proteins) in farmraised fish.


Aggressive experiments starting in the 1990s exposed pigs to BSE-positive material orally, intrathecally and parenterally to determine if single-step transmission of a prion could be established. Although injecting material directly into the brain of the pig could result in signs consistent with spongiform encephalopathies, high-level exposure to contaminated feed did not result in transmission. A number of researchers have explored this species barrier and it appears that transmission of BSE to pigs would be difficult. However, once transmitted to pigs, Liberski and coworkers demonstrated pathology in the brain consistent with BSE, Scrapie and other prion diseases. Findings by Castilla et al. using a mouse model have indicated that perhaps pigs or an intermediate host could transmit prion disease without showing overt signs (subclinical disease). This work has not been fully explored and may not represent a field possibility. Again, although further work is needed to define the risk, there is clearly cause for concern about using animal-derived proteins in farm-raised pigs.

Laboratory Diagnosis

The diagnosis of prion diseases in the lab is unique in that common methodologies such as PCR, serology and cell culture assays are ineffective. This is because prions are a proteinaceous conformer of normal human PrP, lack a nucleic acid component, and do not cause an inflammatory or immunological response due to the host's inability to recognize it as foreign. Furthermore, abnormal prions are unevenly distributed in the host: with the highest concentrations being present in nervous system tissues, and only low concentrations in easily accessible body fluids such as cerebral spinal fluid, blood and urine.

The "gold standard" for the diagnosis of TSEs is through post-mortem neuropathological examinations of brain tissues. Using light microscopy, histological features such as vacuolation, spongiform changes, astrocytic gliosis and amyloid plaques can generally be observed. However, in some cases not all or any of these features may be present.

Immunohistochemistry performed on formalin-fixed or frozen tissue sections of the brain or lymphoid tissue can also be applied to directly detect the presence of [PrP.sup.Sc]. This technique can be extended to tonsil biopsies in patients suspected of having vCJD; yet, the absence of [PrP.sup.Sc] in these tissues is not enough to rule out prion disease.

Other methodologies rely on the biochemical differences in [PrP.sup.C] versus [PrP.sup.Sc]. The idea is that after limited proteolysis using agents such as proteinase K, [PrP.sup.C] is completely hydrolyzed and [PrP.sup.Sc] is reduced to the smaller protease-resistant fragment PrP 27-30. This smaller fragment is then capable of being detected by immunoassays such as Western blots and enzyme imunoassays. A newer methodology called protein misfolding cyclic amplification (PMCA) may prove to be highly useful in peripherally infected individuals, such as those with vCJD, because of its ability to detect prions in the blood.

Predictive genetic testing is also available to help identify if a person is at an increased risk of developing a prion disease prior to the onset of symptoms. However, it is estimated that only 40% of genetic prion cases have a family history associated with them. Typically testing requires only a blood sample, which allows laboratories to find subtle changes in the Prnp gene through sequencing.

Potential Therapies

Currently there are no successful or effective therapies that exist for prion disorders or other neurodegenerative diseases. The most logical therapies for prion disorders would likely be through the direct interaction of small molecules targeted at either blocking the conversion of [PrP.sup.C] to [PrP.sup.Sc], or reversing it. Another strategy for prion therapies would be to develop or discover small molecules that would act more indirectly by effectively modulating proteostasis networks. Once modified, the host would naturally boost cellular pathways by upregulating proteostasis components that could eliminate, inhibit, or refold infectious prions. Since the human body does not produce antibodies against infectious prions, anti-PrP antibody therapy designed at blocking the ability of abnormal [PrP.sup.Sc] to interact with normal [PrP.sup.C] is also under investigation.

Although a number of compounds that inhibit the conversion of [PrP.sup.C] to [PrP.sup.Sc] have already been discovered, and proteostasis modulators are emerging, there are many roadblocks remaining in finding an effective therapy for prion disorders. Most therapies would necessitate a method capable of detecting prion diseases at the earliest stages in order to be effective. However, such a technology does not yet exist. Also, the molecules being used must have a limited toxicity, good bioavailability, and be able to penetrate the blood-brain barrier in order to work effectively in the central nervous system. Drug resistance must now be taken into consideration as studies confirm the ability of prion strains to be susceptible to selection pressures, resulting in drug-resistant strains.

Disinfection and Sterilization

Since the discovery of iCJD, the importance of developing effective disinfection and sterilization methods for the inactivation of prions has emerged. This is especially true for surgical instruments used during procedures where direct contact has taken place with highly infectious tissues such as the brain, spinal cord, pituitary gland, tonsils and posterior eye. Unlike most viruses and bacterial pathogens, prions lack nucleic acids and thus they are not easily inactivated by common techniques such as autoclaving, exposure to UV light, gamma-ray irradiation, and alcohols. They also have been shown to bind tightly to many surfaces and increase in resistance when dried. Therefore, severe physical and chemical conditions are necessary to undergo successful disinfection and sterilization.

Prions in Pathology and Laboratory Medicine

Every laboratory should have a procedure for dealing with tissue and other fluids from patients suspected or confirmed to have CJD or other prion infection.

Working with tissue or fluids from patients with suspected prion disease requires biosafety level 2 (BSL-2) or BSL-3 depending on where the fluid or tissue originated. Unfixed tissues of brain or spinal cord, as well as tissues such as lymph nodes from patients with known disease, have been shown to contain high levels of prions and should be handled at BSL-3. Blood and bone marrow are thought to have only low risk of exposure to prions, even from known cases. Current recommendations indicate that these specimens can be handled safely under BSL-2 conditions by adhering to universal precautions for prevention of transmission of bloodborne pathogens. Following this logic, specimens can be tested in automated analyzers, if instruments are enclosed and waste can be appropriately disposed. Manual processing such as pipetting, decanting, decapping, etc., should be performed within a Biological Safety Cabinet/laminar flow hood. Urine and other fluids are thought to have even less risk than blood and bone marrow, however, prudence would dictate similar precautions to blood.

Personal protective equipment or PPE include barrier protection such as disposable gowns, gloves, and eye protection. In addition to the engineered protection provided by laminar flow containment hoods, use of additional PPE are recommended when handling potentially contaminated specimens.

Since prions are not inactivated completely by common laboratory decontamination products, disposable laboratory equipment should be used whenever possible. Following standard protocols, laboratory waste from suspected prion disease testing as well as all infectious waste, should be terminally incinerated. Treatment with either 6% or greater sodium hypochlorite or > 1N sodium hydroxide for at least 1 hour can effectively deactivate prions and therefore is appropriate for cleaning up spills.

Formalin alone does not inactivate prions, thus in several parts of the laboratory, additional steps need to be taken to inactivate these agents. The College of American Pathologists recommends the following for safe handling of tissue: the sample should be entirely immersed in formic acid for 1 hour, and subsequent formalin fixation for 2 days prior to embedding. Formic acid inactivates formalin-treated prions but has minimal effects on the quality of histology. Disposable equipment such as knives, etc., should be used whenever possible. Cleaning or reprocessing equipment following manipulation of suspected prion tissue materials should be carefully reviewed.

Shipping of materials suspected of containing prions, must comply with the "Recommendations of the United Nations Committee of Experts on the Transport of Dangerous Goods." Prions are listed in category 6 - Toxic and Infectious Substances, Division 2 - Infectious Substances. Appropriate and certified shipping containers and processes must be used.

Table 4: Tissue Preparation for Human CJD and Related Diseases

1. Histology technicians wear gloves, apron, laboratory coat, and face protection.

2. Adequate fixation of small tissue samples (e.g., biopsies) from a patient with suspected prion disease can be followed by post-fixation in 96% absolute formic acid for 30 minutes, followed by 45 hours in fresh 10% formalin.

3. Liquid waste is collected in a 4L waste bottle initially containing 600ml 6N NaOH.

4. Gloves, embedding molds, and all handling materials are disposed as regulated medical waste.

5. Tissue cassettes are processed manually to prevent contamination of tissue processors.

6. Tissues are embedded in a disposable embedding mold. If used, forceps are decontaminated as in Table 10.

7. In preparing sections, gloves are worn, section waste is collected and disposed in a regu lated medical waste receptacle. The knife stage is wiped with 2N NaOH, and the knife used is discarded immediately in a "regulated medical waste sharps" receptacle. Slides are labeled with "CJD Precautions." The sectioned block is sealed with paraffin.

8. Routine staining:

a. slides are processed by hand;

b. reagents are prepared in 100ml disposable specimen cups;

c. after placing the cover slip on, slides are decontaminated by soaking them for 1 hour in 2N NaOH;

d. slides are labeled as "Infectious-CJD."

9. Other suggestions:

a. disposable specimen cups or slide mailers may be used for reagents;

b. slides for immunocytochemistry may be processed in disposable Petri dishes;

c. equipment is decontaminated as described above or disposed as regulated medical waste.

Taken from CDC/NIH BMBL 5th ed, Section III: Laboratory Biosafety Level Criteria.

Table 5: Prion Inactivation Methods for Reusable Instruments and Surfaces

1. Immerse in 1N NaOH, heat in a gravity displacement autoclave at 121[degrees]C for 30 minutes. Clean and sterilize by conventional means.

2. Immerse in 1N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Transfer into water and autoclave (gravity displacement) at 121[degrees]C for 1 hour. Clean and sterilize by conventional means.

3. Immerse in 1N NaOH or sodium hypochlorite (20,000) for hour. Rinse instruments with water, transfer to open pan and autoclave at 121[degrees]C (gravity displacement) or 134[degrees]C (porous load) for 1 hour. Clean and sterilize by conventional means.

4. Surfaces or heat-sensitive instruments can be treated with 2N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Ensure surfaces remain wet for entire period, then rinse well with water. Before chemical treatment, it is strongly recommended that gross contamination of surfaces be reduced because the presence of excess organic material will reduce the strength of either NaOH or sodium hypochlorite solutions.

5. Environ LpH (EPA Reg. No. 1043-118) may be used on washable, hard, non-porous surfaces (such as floors, tables, equipment, and counters), items (such as nondisposable instruments, sharps, and sharp containers), and/or laboratory waste solutions (such as formalin or other liquids).

CDC Note: This product is currently being used under FIFRA Section 18 exemptions in a number of states. Users should consult with the state environmental protection office prior to use.

Taken from CDC/NIH BMBL 5th ed, Section III: Laboratory Biosafety Level Criteria


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Table 1

Year Primary host Disease Disease Forms/
reported Transmission

1732 Sheep and goats Scrapie Not
 transmitted to

1920 Humans Creutzfedlt-Jakob Familial,
 Disease sporadic and
 human to

1928 Humans Gerstmann-Strassler- Familial
 Scheinker disease

1941 Humans Kuru Human to human

1947 Mink Transmissible Mink Not
 Encephalopathy transmitted to

1967 Elk and Deer Chronic Wasting No
 Disease demonstrated
 to man

1986 Humans Fatal Familial Familial
 Insomnia disease

1986 Cattle Bovine Spongiform Transmission
 Encephalopathy to humans

1986 Antelope, Bison Exotic Ungulate No
 Spongiform demonstrated
 Encephalopathy transmission
 to man

1990 Domestic cats, Feline Spongiform No
 large cats in Encephalopathy demonstrated
 captivity transmission
 to man

1995 Humans New Variant Human
 Creutzfedlt-Jakob transmission
 Disease demonstrated

1996 Primates in Zoo Primate Spongiform No
 captivity Encephalopathy demonstrated
 to man

Table 2: Sterilization Methods for Prions

Effective Ineffective

Autoclave at Standard autoclaving
121[degrees]C-132[degrees]C for 1 methods
hour (gravity displacement (121[degrees]C for
sterilization) 15 minutes)

Autoclave at 121[degrees]C for 30 Dry heat
minutes (prevacuum sterilization)

Autoclave at 134[degrees]C for 18 Boiling
minutes (prevacuum sterilization)

Autoclave at 134[degrees]C for 18 Ethylene oxide
minutes immersed in water

Sodium dodecyl sulfate 2%, plus Formaldehyde
acetic acid 1%, plus autoclave at
121[degrees]C for 15-30 minutes

0.09 N or 0.9 N NaOH for 2 hours, Hydrogen peroxide gas
plus autoclaved at 121[degrees]C for plasma only
1 hour (gravity displacement

Vaporized hydrogen peroxide (1.5-2 Ionizing radiation
mg/L, 30[degrees]C, 3 cycles)

Radiofrequency gas plasma Microwave

Enzymatic detergent (0.8%, UV light
43[degrees]C for 5 minutes), plus
hydrogen peroxide gas plasma
sterilization (1.5 mg/L,
25[degrees]C for 3 hours)

Table 3: Chemical Inactivation of Prions

Effective Ineffective

Alkaline and Alkaline and Enzymatic detergents
detergents* and a
1.6% Alkaline
(43[degrees]C /
15 min)

Chlorine 50 ppm Chlorine dioxide
(>1,000 ppm)

59% Hydrogen 0.2% - 60% Hydrogen peroxide

0.2% Peracetic 0.2% - 19% Paracetic acid

Phenolic Acetone
and a 5% Phenolic
(20[degrees]C /
30 min)

[greater than or 50% - 100% Alcohol
equal to] 1 N NaOH
(20[degrees]C for
1 hour)

0.01 M Sodium 1.0 M Ammonia

3% Sodium dodecyl 3.7% Formaldehyde
(100[degrees]C /
10 min)

0.5 mmol/L Copper 2% Iodine
and 100 mmol/L
hydrogen peroxide

20,000 ppm NaOCl 5% Glutaraldehyde
(20[degrees]C / 1

Quaternary Quaternary ammonium compound
ammonium compound (specific formulation)

3 M guanidine 1.0 N Hydrochloric acid
thiocynate (RT / 2

3 M 0.55% Ortho-phthalaldehyde
acid (RT / 2

7 M guanidine 1% - 5% Sodium dodecyl sulfate
hydrochloride (RT
/ 2 hours)

60% formic acid 5% Sodium deoxycholate
(RT / 2 hours)

50% phenol (RT for Phenols/phenolics
/ hours)

 5% Tego

 1% Triton X-100

 4-8 M Urea

* Specific formulations: Klenzyme (Steris), Hamo-100
(Steris), Septo-Clean (Septo-Clean), CIP100 (Steris),
Prionzyme-M (Genecor), and Rely-On (DuPont).

** Contains: 6.4% ortho-benzyl-para-chlorophenol, 3%
para-tertiary-amylphenol, 0.5% ortho-phenyl phenol,
4.9% hexylene glycol, 12.6% glycolic acid, and 8%
isopropanol. NOTE: Some chemical processes may be
listed as both effective and ineffective based on
differences in testing methodologies during studies
including: strain of prion tested, pH, exposure times,
temperature differences, and chemical concentrations.

* Matthew Henson, BS, MLS(ASCP)[CM], Medical Laboratory Scientist, Diagnostic Infectious Testing Laboratories, Department of Pathology and Laboratory Medicine, Cincinnati Children's Hospital

Mark Ireton, MA, BS, MLS(ASCP)[CM], Program Coordinator, Medical Laboratory Science Program, University of Cincinnati

Joel E. Mortensen, PhD., HLCD (ABB), MLT (AMT), CLC (AMT), Director, Diagnostic Infectious Diseases Laboratory, Cincinnati Children's Hospital, Cincinnati, Ohio
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