MicrobiologyBytes

A decade ago, a new variant form of Creutzfeldt-Jakob disease was identified. The emergence of this prion disease in humans was the consequence of the zoonotic transmission of bovine spongiform encephalopathy through dietary exposure. Since then, the control of human exposure to prions has become a priority, and a policy based on the exclusion of known infectious materials from the food chain has been implemented. Because all investigations carried out failed to reveal evidence of infectivity in milk from affected ruminants, this product has continuously been considered as safe. In this study, researchers demonstrate the presence of prions in colostrum and milk from sheep incubating natural scrapie and displaying apparently healthy mammary glands. This finding indicates that milk from small ruminants could contribute to the transmission of prion disease between animals. It also raises some concern with regard to the risk to humans associated with milk products from ovine and other dairy species.

Prions in Milk from Ewes Incubating Natural Scrapie. 2008 PLoS Pathog 4(12): e1000238
Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.

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Researchers have found novel prion infectivity in white and brown fat tissues of mice. Prion diseases, also known as transmissible spongiform encephalopathies, are infectious progressive fatal neurodegenerative diseases which affect humans as well as wild and domestic animals. Distribution of prion infectivity in organs and tissues is important in understanding prion disease pathogenesis and designing strategies to prevent prion infection in animals and humans. Previous studies in animals including sheep, goats, cattle, deer, mink, hamsters and mice, have found prion infectivity mostly in nervous system tissues such as the brain and spinal cord. The tissues studied in a mouse model demonstrate a proof of principle that white and brown fat tissues are sites of prion agent deposition and therefore may play a previously unrecognized role in prion infectivity and transmission of prion disease. The authors state clearly that it will be important to extend their studies to prion-infected large animals, such as cattle, sheep, deer, and elk where they may be potential sources of contamination of human and domestic animal food chains. Results of the current and future studies may merit additional consideration of steps to eliminate from the food chain any fat from ruminants suspected of exposure to or infection with prions.

Detection of Prion Infectivity in Fat Tissues of Scrapie-Infected Mice. 2008 PLoS Pathog 4 (12): e1000232
Distribution of prion infectivity in organs and tissues is important in understanding prion disease pathogenesis and designing strategies to prevent prion infection in animals and humans. Transmission of prion disease from cattle to humans resulted in banning human consumption of ruminant nervous system and certain other tissues. In the present study, we surveyed tissue distribution of prion infectivity in mice with prion disease. We show for the first time detection of infectivity in white and brown fat. Since high amounts of ruminant fat are consumed by humans and also incorporated into animal feed, fat-containing tissues may pose a previously unappreciated hazard for spread of prion infection.

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• MicrobiologyBytes: Prions
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• How Now Mad Cow

The mother of a Spanish man who died from the human form of mad cow disease has also died from the illness. The mother, in her early 60s, died in August 2008. Her son, 41, died in February 2008. Three of these cases in Spain were in the same northern province, Leon. Researchers will try to determine whether the mother and son shared a genetic background making them more susceptible to contracting the illness.
The first confirmed death from mad cow disease in Spain was in 2005, when a young woman died near Madrid. In addition to the cases in Spain, as of June 2008 the following numbers of cases of human mad cow disease had been reported: 167 in the United Kingdom; 23 in France; 4 in Ireland; 3 in the United States; 2 each in Netherlands and Portugal; and one each in Canada, Italy, Japan and Saudi Arabia.

CNN

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• Prion Diseases
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Researchers from the United States Department of Agriculture have identified a novel mutation in the bovine prion protein gene in a cow confirmed with atypical Bovine Spongiform Encephalopathy (BSE). This is the first report of a confirmed case of BSE (aka mad cow disease) with a potential pathogenic mutation within the bovine Prnp gene. BSE, a transmissible spongiform encephalopathy (TSE) or prion disease of cattle, was first discovered in the United Kingdom in 1986. BSE is considered to be the cause of a human prion disease known as variant Creutzfeldt-Jakob Disease (vCJD). Veterinary scientists have now identified a novel mutation, E211K, in the bovine Prnp gene. This mutation is identical to the E200K pathogenic mutation in the human Prnp, which has been described as the most common cause of genetic CJD. The study supports the view that all three etiological forms of TSEs in humans are also present in cattle: infectious, sporadic, and genetic. It further supports the hypothesis that the BSE epidemic may have originated from a genetic case of cattle BSE. Cattle with similar mutations can be expected in cattle herds world-wide and could be the source of new BSE outbreaks. It is therefore critical to continue world-wide surveillance for typical and atypical BSE cases including sequencing of the Prnp gene. A newly developed assay system for detecting the E211K mutation has been developed for this purpose. Finally, in order to protect humans it is essential to continue to exclude Specified Risk Materials from the food chain and to maintain the ruminant feed ban.

BSE Case Associated with Prion Protein Gene Mutation. PLoS Pathog 4(9): e1000156
Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD) in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp). Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K) in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000) in cattle. We hypothesize that the bovine Prnp E211K mutation most likely has caused BSE in the approximately 10-year-old cow carrying the E221K mutation.

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The origin of the transmissible agent involved in the foodborne epidemic of bovine spongiform encephalopathy (BSE) remains a mystery. It has recently been proposed that this could have been the result of the recycling of an atypical, more probably sporadic, form of BSE (called bovine amyloidotic spongiform encephalopathy, or L-type BSE) in an intermediate host, such as sheep. A team from the French Food Safety Agency has identified a prion protein characteristic that is unique to some natural but unusual sheep scrapie cases. This finding may provide a novel method by which to study prion diversity and their possible changes during cross-species transmission. Mystery still surrounds the origin of the transmissible agent involved in the food-borne epidemic of bovine spongiform encephalopathy (BSE). Classical BSE, more commonly known as mad cow disease, is a known cause of a variant form of the incurable, degenerative neurological disorder Creutzfeldt-Jakob disease in humans. It has recently been proposed that this could have been the result of the recycling of an atypical, more probably sporadic form of BSE (called bovine amyloidotic spongiform encephalopathy, or L-type BSE) in an intermediate host, such as sheep. The team analyzed the molecular features of the disease-associated protease-resistant prion protein (PrPres) to determine any differences which might discriminate between scrapie and BSE cases. The researchers sampled PrPres from the brains of transgenic mice overexpressing the ovine prion protein after experimental infection with prions from bovine classical BSE, L-type BSE, and ovine scrapie. Scrapie cases were found to include rare “CH1641-Like” isolates, which share some PrPres molecular features with classical BSE and L-type BSE. The molecular features of the prion protein in the “CH1641-like” sheep scrapie cases more closely resemble those found in L-type BSE compared to classical BSE. However, from a series of four “CH1641-like” scrapie cases, the researchers found a pathological C-terminal prion protein product that was undetectable from both L-type and classical BSE transmitted to such mice, clearly suggesting that such scrapie isolates are not linked to these BSE forms. Further studies to confirm this discriminating factor are needed in sheep, especially from sheep experimentally infected with L-type BSE, which were not available for this study. These findings add a novel approach for the discrimination of prions that may help to understand their possible changes during cross-species transmissions.

A C-Terminal Protease-Resistant Prion Fragment Distinguishes Ovine “CH1641-Like” Scrapie from Bovine Classical and L-Type BSE in Ovine Transgenic Mice. 2008 PLoS Pathog 4(8): e1000137
The protease-resistant prion protein (PrPres) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrPres in four “CH1641-like” natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if “CH1641-like” isolates might be linked to L-type BSE. We found less diglycosylated PrPres than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrPres form (PrPres #1) to L-type BSE. However, the “CH1641-like” isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrPres product (PrPres #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrPres #1 and PrPres #2 resulted in enrichment in PrPres #2, and demonstrated the presence of mono- and diglycosylated PrPres products. PrPres #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrPres #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between “CH1641-like” ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.

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• New forms of bovine spongiform encephalopathy
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Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrP(C)) to a misfolded, pathogenic isoform (PrP(Sc)). Prion inoculation experiments in mice expressing homologous PrP(C) molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, researchers analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. They tested 20 candidate genes whose products either colocalize with PrP, are associated with Alzheimer’s disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrP(C), the data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta precursor protein (App) or interleukin-1 receptor type I, and transgenic overexpression of human superoxide dismutase 1 prolonged incubation times by 13, 16 and 19%, respectively. Clearly the conversion of PrPC to PrPSc involves a complex pathway, the elucidation of which would benefit treatment of these devastating diseases.

Genes contributing to prion pathogenesis. J Gen Virol 89 2008, 1777-1788

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BBC News: At least 35,000 people have taken part in the latest mass protest in South Korea against the government’s decision to allow US beef imports to resume. South Korea used to be a big market for US beef, but suspended imports in 2003 after a BSE (“mad cow disease”) outbreak. The decision to lift the ban has led to weeks of protests based on health fears and discontent with the government. As a result, the South Korean government has sought extra safety assurances from the US authorities on American beef. Both governments insist the beef is safe.

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Prions are proteinaceous infectious agents that were first discovered because of their role in the etiology of transmissible spongiform encephalopathies (TSEs), a set of fatal neurological disorders that include Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalitis. TSEs arise from progressive misfolding of the endogenous cellular prion protein (Prp [PrPC]) into disease-associated scrapie form (PrPSc), which, in turn, disrupts normal cellular function and results in the formation of aggregates and amyloid-like plaques. Although there has been a clear association between PrPSc with these disease states, the cellular function of PrPC remains incompletely understood. PrPC is expressed across the entire central nervous system and at particularly high levels in the hippocampus, striatum, and frontal cortex, with apparently wide subcellular distribution, including synaptic sites. A synaptic role of PrPC is consistent with evidence from PrP-null mice showing deficits in spatial learning, altered long-term potentiation and increased excitability of hippocampal neurons. Several studies have also suggested that PrPC may provide neuroprotection. For example, cultured hippocampal neurons obtained from PrP-null mice show an increased apoptosis during oxidative stress. Mice lacking PrPC show increased neuronal damage after ischemic stroke, whereas protection is evident upon the viral-based overexpression of PrPC in rats. Finally, in several in vivo models of seizure activity, PrP-null mice showed increased mortality, likely as a consequence of hyperexcitability leading to excitotoxicity. The cellular and molecular basis for these effects remains unknown. Recent research shows that PrPC exerts a neuroprotective role by inhibiting excitotoxic cell death and that PrPC is a modulator of synaptic function and, consequently, neuronal excitability.

So that’s what prions are for!

Prion protein attenuates excitotoxicity by inhibiting NMDA receptors. J Cell Biol 2008 181: 551-565
It is well established that misfolded forms of cellular prion protein (PrP [PrPC]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrPC remains incompletely understood. To determine the physiological role of PrPC, we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-D-aspartate (NMDA)–evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrPC. The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrPC mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits.

The amyloidoses are a group of protein misfolding disorders characterized by the accumulation of amyloid fibrils formed from a variety of proteins that, under normal physiological conditions, are harmless and soluble. Currently, 25 amyloid diseases have been identified, such as the prion diseases, Alzheimer’s disease, type 2 diabetes, and various systematic amyloidoses. Although the various proteins that can polymerize into amyloid fibrils have unrelated sequences, they can all form fibrils with a similar ultrastructural appearance. Among them, prion diseases such as transmissible spongiform encephalopathy (TSE), including scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) of deer and elk, are highly infectious. In these diseases, prion (PrPSc), an abnormal form of the host cellular prion protein (PrPC), induces the conformational change of PrPC to the PrPSc and causes a detectable phenotype or disease in the affected individual. AA amyloidosis is characterized by the systemic deposition of extracellular fibrils composed of amyloid A protein, primarily in the spleen; liver; and, to a lesser extent, in other organs. In most species, AA amyloidosis occurs sporadically and is typically secondary to chronic inflammation, infection, or neoplasia. Intriguing recent data suggest that AA amyloidosis could be transmitted by a prion-like infectious process through a seeding-nucleation mechanism.

The cheetah (Acinonyx jubatus) is in danger of extinction and is included on The World Conservation Union list of vulnerable species. Although efforts have been made in wildlife sanctuary parks and zoos worldwide to prevent extinction, a steady increase in the size of the cheetah population is hampered by the high prevalence of certain diseases in captive cheetahs. In particular, systemic AA amyloidosis is regarded as an increasingly important cause of morbidity and mortality in captive cheetahs as prevalence increased from 20% in pre-1990 reported necropsies to an unusual 70% of necropsied cheetahs in 1995. Despite much effort, the pathogenesis for AA amyloidosis in cheetahs is still only partially understood. However, environmental epidemiological studies indicate that breeding conditions have a prominent effect on the incidence of AA amyloidosis. A high rearing density is always associated with early age of onset, and with the high incidence and severity of AA amyloidosis, findings similar to sheep scrapie and cervid CWD. Thus, sustained epidemics of sheep scrapie and cervid CWD appear to be principally due to horizontal (animal to animal) transmission, although the routes of natural transmission remain to be clarified. The propagation of AA amyloidosis among captive cheetah populations may also depend on a horizontal transmission pathway. Identification of the mode of transmission is a prerequisite for disease control. This study shows that the faeces from a cheetah with AA amyloidosis can act as a possible transmission agent to accelerate the onset of AA amyloidosis.

Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease PNAS USA May 12, 2008
AA amyloidosis is one of the principal causes of morbidity and mortality in captive cheetahs (Acinonyx jubatus), which are in danger of extinction, but little is known about the underlying mechanisms. Given the transmissible characteristics of AA amyloidosis, transmission between captive cheetahs may be a possible mechanism involved in the high incidence of AA amyloidosis. In this study of animals with AA amyloidosis, we found that cheetah feces contained AA amyloid fibrils that were different from those of the liver with regard to molecular weight and shape and had greater transmissibility. The infectious activity of fecal AA amyloid fibrils was reduced or abolished by the protein denaturants 6M guanidine HCl and formic acid or by AA immunodepletion. Thus, we propose that feces are a vehicle of transmission that may accelerate AA amyloidosis in captive cheetah populations. These results provide a pathogenesis for AA amyloidosis and suggest possible measures for rescuing cheetahs from extinction.

• The first-ever cases of chronic wasting disease (CWD) in wild elk have been discovered in Canada.

But wait -

It might seem like prions are always bad news, but if that were true, they would be an evolutionary disadvantage and would surely have been lost. So the truth must be more complicated, and in tomorrow’s post, we’ll see how.

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