Dr. Marguerite Pappaioanou is associate director of science and policy for the Office of Global Health at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA. Both a veterinarian and epidemiologist, Dr. Pappaioanou has maintained a strong interest in international and domestic public health, especially in those areas where human and veterinary medicine intersect.

Overview

The presentation is going to be a general overview of transmissible spongiform encephalopathies. We will cover the following issues of prion diseases.

• Different TSEs
   
• Biology of TSEs and neuropathological characteristics
   
• Transmission of TSEs
   
• Species barriers and species jumping
   
• BSE and vCJD as specific examples
   
• Monitoring of BSE and CJD in the US
   
• Prevention and control of TSEs

Transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies are neurodegenerative diseases in animals and humans. They are characterized by long incubation periods that are relative to the host species infected. The lesions are primarily restricted to the central nervous system. The disease can cause dementia, ataxia, insomnia, paraplegia, parasthesias, and deviant behavior. The outcome of these diseases is inevitably fatal; there is no treatment or cure for these diseases. The causative agent for TSEs are prions (proteinaceous infectious particles), which are abnormal infectious proteins that are not connected with nucleic acid structures.

This slide shows the two groups of TSE: human TSEs and animal TSEs.

[Figure:   Human TSEs and animal TSEs]

This table lists the TSEs for humans and animals as well as the main host species, how the disease is transmitted and the primary geographic location for the disease.

[Figure:   TSEs]

Human TSEs

The most well known human TSE is Creutzfeld Jacob disease (CJD) which is a sporadically occurring disease. CJD accounts for 85-90% of the human TSEs and is more common in the elderly population. Variant CJD is related to bovine spongiform encephalopathy (BSE). The other human TSEs include kuru, which was related to cannibal practices among the Fore tribe of Papua New Guinea. When those practices were abandoned, kuru also disappeared.

Human TSEs can occur in 3 ways: sporadic, inherited and acquired. Spontaneous CJD occurs one case per million persons and the mechanism is unknown but may be a somatic mutation or spontaneous conversion. Inherited or familial forms (GSS) can pass from parent to child and is thought to be a germ line mutation in the PrP gene. The inherited form accounts for 10-15% of the TSEs. Acquired forms such as variant CJD is thought to be through oral exposure (infected meat), iatrogenic (dura mater transplants) or possibly through contaminated instruments or blood. This form is much less common and is thought to account for 1% of the human TSEs. New variant CJD differs by presenting at an early age (average 28 years), has a longer duration of illness (13 months). It present with more psychiatric and sensory signs and has delayed neurologic findings. Infectivity has been detected in lymphoid organs such as tonsils, lymph nodes and spleen.

For these diseases we cannot fulfill Koch’s postulates for disease transmission since we are not dealing with a virus or bacteria.

Animal TSEs

Here is a list of the common animal TSEs with pictures of the presentation of the disease.

[Figure:   Animal TSEs]

Scrapie

The most well known animal TSE is scrapie, which affects sheep and goats. Written documentation for scrapie dates back to 1732. It is a fatal, degenerative disease affecting the central nervous system of sheep and goats. Only sheep that have a specific genotype and then are exposed to the infectious agent appear to be affected by the disease. The interaction between the scrapie agent and host genetic mechanism is not well understood. To date, scrapie has not been found to be infectious to humans.

This map shows the spread of scrapie between 1938 and 1977.

[Figure:   Spread of Scrapie, 1938 - 1977]

Note that Australia and New Zealand were affected but they are now scrapie free.

Chronic Wasting Disease

Chronic wasting disease in deer and elk was first noted in captive deer in a research facility in Colorado in the 1960s. The disease primarily affects the central nervous system but can also be detected in tonsils, oropharynx and intestinal lymphoid tissues. Its route of infection is oral, also possible by direct interaction with feces, urine, saliva, and placenta. Environmental contamination in pastures with decomposing carcasses and scavenging is another possible route. The agent can spread before any clinical signs appear.

This North American map shows the primary geographic distribution of CWD in North America.

[Figure:   Reported distribution of CWD in North America, 1981-2002]

Here are pictures comparing normal and CWD-affected brain tissue:

[Figure:   deer brain tissue normal]

[Figure:   deer brain tissue CWD]

[Figure:   deer brain tissue normal and CWD]

Feline Spongiform Encephalopathy

This was the first instance where scientists suspected that a prion could jump species. There were cases where both domestic and wild cats in zoos developed the disease. Epidemiology studies showed the cats were being fed with material feed from cattle.

Transmissible Mink Encephalopathy

This was thought to come from scrapie and jumped species in some way that is not yet clear.

Neuropathologic Characteristics and Biology of Prions and TSEs

The following are the neuropathologic characteristics of TSEs seen on brain specimen

• Absence of wide spread atrophy
   
• Minimal to widespread neuronal loss
   
• Sparse to widespread vacuolation or spongiform changes
   
• Mild to severe reactive astrocytic gliosis
   
• Absence to abundance of PrP amyloid plaques (pathognomonic for TSE)
   

The biology of prion disease is felt to be a change of the normal prion structure. Host protein (PrP) is expressed in many body tissues but the highest levels are in the brain. The brain has 50% more prions than other tissues. As we develop prions they are also phagocytized to keep them at a normal number. It is not clear what all the reasons are for prion existence. However, they may play an important functional role in nerve impulses being transmitted. In non-neuronal cells, PrPc is found at cell membrane. In neuronal cells, PrPc presents at pre-synaptic nerve terminals. This plays a role in recycling of synaptic vesicles or a more direct role in synaptic transmission.

Prions are made up of about 200 amino acids. They are of a 3 helix structure.

Here is a normal bovine prion protein with the 3-helix structures.

[Figure:   Bovine Prion Protein]

There are 3 helixes which are designated as the alpha region, and they have 2 beta strands which have a more flattened appearance.

The prion disease is thought to be due to abnormally folded protein. A normal protein has a certain structure and configuration. Somehow they become abnormal and folded when the disease is present. How and why this happens is unknown. The terminology used for an abnormal folding is PrPc2 variations including PrPsc (from scrapie) and Pr Pres (for residual). These abnormal prions appear to be encoded by the host gene. They are protease resistant making it difficult to destroy. Up to 40 % of the amino acid chain is in the ? sheet conformation.

TSEs result from accumulation of the PrPsc in the brain. They are the only known infectious pathogens thought to be devoid of nucleic acid. PrPsc can have a variety of conformations; each is associated with a specific disease. They are also not able to be destroyed by cooking, autoclave, enzymes or disinfection.

This slide shows the normal folded protein and what happens when it is affected by the abnormal prion.

[Figure:   Normal Protein and Prion forms]

The mechanism for transformation is unknown. However, when a normal form comes into contact with an abnormal form it is changed into the abnormal. The abnormal form is also not susceptible to phagocytosis or lysis thereby letting the numbers increase in the brain. The proteins abnormal conformation replicates and accumulates to the point of clogging neurons. The neurons then die from apoptosis which leads to the vacuoles as seen on previous slides.

Strains of TSEs

There appear to be different biological and biochemical characterizations for the different TSEs strains. This is based on several differences: length of incubation time, distribution of the vacuoles, severity of the spongiform changes, resistance to chemical and heat inactivation.

Theory 1: PrP is associated with an unidentified informational molecule--possibly nucleic acid in nature.

Theory 2: PrPsc can have a variety of conformations, each associated with specific disease, i.e. strains. It is this theory that the experts are more strongly leaning toward.

Transmission of TSEs

Naturally occurring TSEs are usually associated with individual species (i.e scrapie—sheep, goats; TME—mink; sporadic CJD—humans, CWD—elk, deer). All humans infected with variant CJD have two alleles of codon 129 or PrP carrying methionine. They are homologous for methionine at this codon. Only 30% of people have this homology. Susceptibility of prion infection increases with matched donor and recipient PrP sequences. Differences in amino acid sequence of PrP between species reduces the efficiency of prion transmission, which would make it less able to jump species.

Species barriers/species jumping

Species barriers may be absolute where the prion is incapable of being infectious to another species, it may be partial where only a proportion of animals will be affected on first passage, or it may result in extended incubation period on first passage as we see with BSE and vCJD.

In non-refractory species the variations are attributed to:

• The origin of the prion/TSE agent
   
• The PrP genotype of the recipient
   
• The route of infection
   
• The dose of infective tissues
   
• Possibly the age of the exposed
   

Some problems with this however, is that species barrier efficacy cannot be predicted. The relative impregnability of a barrier depends on differences in the PrP amino acid sequence between species and on the particular strain of prions involved. This theory is not well understood. An experiment to determine susceptibility of cattle to CWD by oral challenge is currently in progress.

BSE and vCJD

BSE in cattle was first recognized in 1986. By 1992 there were over 36,700 cases documented. The incubation period is thought to be 4-8 years. The original cause theory is that the sheep scrapie was transformed in cattle. A second theory is that the condition occurred because of changes in feeding practices and was spread in this manner. Still unanswered is how it is transferred from cow to cow: through feces? long term soil contamination? Placenta? Vertical transmission?

In 1980-81 there were changes in the rendering industry practices in the United Kingdom. Previously there was use of organic hydrocarbon solvents for several hours at high heat and a steam process to remove the solvent which allowed the extraction of tallow and other substances. Due to cost of production and decrease in a market for tallow the rendering process was changes and did not include these steps. It is suspected that the agent was not inactivated using the new method and was incorporated into cattle feed in form of bovine meat and bone meal.

The link between BSE and vCJD (human TSE) is suspected on several levels. First, the age at clinical onset of the disease is much younger than sporadic TSE and indicates likely period of exposure. Similarities in appearance of PrPsc patterns have been demonstrated using Western immunoblotting. There are similarities in lesion profiles induced in mice and macaques. There is circumstantial epidemiological evidence from the geographical and temporal occurrence of the two diseases. In the UK, 182,403 infected cattle and 145 human cases have been noted since 1987. The potential source of human BSE is as yet unknown but the most likely theory is through contaminated food/cattle tissue consumption. Other possibilities include direct contact to affected cattle, but no cases in humans to date have fulfilled this theory. Contact with specific products containing infected materials is also possible, but there is no common exposure date.

Here is a picture of a cow with possible sources of infectivity sites.

[Figure:   Distribution of Infectivity]

Spinal cord, dorsal root ganglion, bone marrow, brain and ileum are all thought to be potential sources of contamination. Milk and skeletal muscle are thought to be at low risk for contamination.

Here are graphs of cases of BSE in UK:

[Figure:   Confirmed cases of BSE in UK]

[Figure:   Confirmed cases of vCJD in UK]

[Figure:   BSE and vCJD in the UK combined]

Prevention and Control of TSE agents

Excluding TSE agents is challenging as there is no immune response from the host, there is no live animal screening process test yet available (current testing is done on downer cows and brain tissue), screening and surveillance programs vary from country to country, and there can also be spontaneous BSE not related to food contamination. A negative test also does not guarantee absence of infectivity.

Here are the current prevention practices:

• Ban on animal feed containing rendered beef
   
• Ban on feeding meat and bone meal to farm animals
   
• Ban on feeding ruminant-derived meat and bone meal to poultry
   
• Ban on meat from animals older than 30 months of age and certain products from the human food chain
   
• Stricter surveillance
   
• Identification of cattle population that allows trace-backs and trace-forwarding of animals
   

This graph shows the confirmation of BSE cases in UK with the prevention measures put into place.

[Figure:   Confirmed cases of BSE in UK 1980-1998]

Many measures came after the highest number of cases had already occurred.

Preventing and Controlling BSE in the US:

• Feed ban in place since 1988 but strictly enforced ban on rendered beef in place since 1996 in UK
   
• In 1989, USDA banned importation of live cattle and certain cattle products from UK and other BSE endemic countries
   
• In 1997, USDA prohibited importation of cattle and most ruminant products from all European countries, Japan and Israel
   
• In 1997, USDA trains veterinarians and laboratory workers on the clinical and pathologic manifestations of BSE: instituted an ongoing BSE surveillance program
   
• In 1997, FDA prohibits use of most mammalian protein, particularly ruminant tissues, in the manufacture of ruminant feed. (exempt are milk, blood, swine, equine and gelatin)
   
• In 2000, restrictions on all rendered animal protein regardless of species
   
• Slaughtered cattle in US comes mainly from animals <30 months of age
   
• Surveillance program in place since 1990
   
• Tracebacks of cattle imported into the US from UK and Ireland 1981-1989
   
• BSE becomes a notifiable disease
   

This graph shows the number of BSE samples from May 1990 to Sept 2003 in the US.

[Figure:   BSE samples May 1990 - Sept 2003, US]

In the US, 35 million animals were slaughtered in 2003 but only 20,000 tested. It costs $100 per test for prion in brain tissue, $25 per test for rapid antibody test using ground brain tissue. In the US, if every animal were to be slaughtered, it would be $1.8 billion.

Global BSE

This table shows, by country, the number of BSE cases by year.

[Figure:  BSE cases by country]

This global map shows the countries where at least one confirmed BSE case has been reported.

[Figure:   Geographical distribution of countries that reported at least one BSE confirmed case from 1988 to 9 January 2004]

The US at the time of this map had not had a case of BSE, but in December 2003 there was a 4˝ year old downer cow identified as positive for BSE. This was confirmed and traceback showed the cow was imported from Canada in 2001. Selective depopulation and destruction of cattle from the herd in Washington was initiated.

Additional safeguards initiated by USDA and FSIS in December 2003 include:

• Banning of downer (non-ambulatory) cows from the human food supply
   
• Specified risk material and tissues banned from human food chain (i.e. skull, nerve, eyes, spinal cord, tonsils and distal ileum)
   
• Normal cattle targeted for BSE surveillance testing at slaughter, no longer marked “inspected and passed” until confirmation of negative test for BSE
   
• Air infection stunning of cattle is prohibited (to prevent brain tissue from dislocating into tissues of the carcass)
   
• Mechanically separated meat in human food prohibited
   
• Verifiable system of national animal identification immediately implemented
   
• Ban use of dead or disabled cows in the products it regulates
   
• Prohibits mammalian blood and blood products to be fed to ruminants as a protein source
   
• Ban use of poultry litter (bedding, spilled feed, feathers)
   
• Ban use of “plate waste” as feed ingredient for ruminants
   
• Requires equipment, facilities, or production lines to be dedicated to non-ruminant animal feeds if they use protein prohibited in ruminant feed
   

Still unanswered questions exist with regard to these diseases:

• The etiologic agent of scrapie has still not been fully characterized
   
• All the routes of transmission are still unknown
   
• What is the exact pathogenesis of TSEs?
   
• How many cases of vCJD will develop in the US?
   
• How do different strains replicated independently in a single host producing only one type of PrP?
   
• What is the infectious dose necessary for disease? What are the genetic predispositions? Is both vertical and horizontal transmission possible?
   

As you can see we have far more unanswered questions than we have answers for. There is ongoing research into this area and perhaps we will be able to better define this very interesting constellation of diseases. Thank you.

	Go to  Readings

 

© 2004 University of Washington Department of Health Services Box 357660, Seattle, WA 98195-7660 e-mail: carrieho@u.washington.edu