Research Project: Mitigating the Risk of Transmission and Environmental
Contamination of Transmissible Spongiform Encephalopathies Location: Animal
Diseases Research
2013 Annual Report
1a.Objectives (from AD-416): Objective 1: Determine whether goats are a
transmission reservoir for ovine scrapie by developing and validating diagnostic
methods for detecting goat scrapie. Determine the genetic predisposition and
transmission route(s) of goat scrapie.
Subobjective 1.1: Improve eradication efforts by developing improved
methods for antemortem scrapie diagnosis.
Subobjective 1.2: Determine if placenta and milk from goats are potential
sources of scrapie to sheep.
Objective 2: Develop methods to mitigate infectivity of soil-associated
prions by screening soil microbes for potential candidates for bioremediation.
1b.Approach (from AD-416): Scrapie is a complex and rare disorder affecting
outbred farm animals held under a wide variety of husbandry conditions and
exposed to an agent for which the transmissible and pathogenic events remain
largely unknown (125). The work described in the research plan is an extension
of the previous highly productive studies by this research group, addressing the
need for implementation of federal regulations based on the best available
science, often in the face of relatively small sample numbers in the natural
host. The work includes development of specific management and diagnostic tools
and is presented as an integrated series of research objectives. This approach
was selected over a hypothesis based approach. After consulting Glass and Hall
(46), the group determined that the work presented in the following plan was
best represented by goal statements rather than hypotheses because the work
increases the density of data necessary for progress (103) and for support of
current and proposed federal regulations. This project addresses only scrapie,
the TSE of sheep and goats. Chronic wasting disease (CWD) is the TSE of North
America cervids (deer and elk). No live animal work with CWD is included in this
project plan since CWD is not endemic in Washington State, the disease appears
to be highly communicable, the modes of transmission are unknown, and we do not
have suitable biocontainment facilities to conduct CWD studies in large animals.
3.Progress Report: Progress was made on characterizing caprine scrapie in
sheep and goats through continued monitoring and data collection from natural
and experimental cases of sheep-origin or goat-origin scrapie. Tissues were
banked from postmortem examinations on animals developing terminal disease for
use in an origin/strain discrimination study. A study demonstrating the
infectious nature of the goat placenta to goats and sheep through oral exposure
at birth contributed to a better understanding of typical modes of transmission
in mixed sheep and goat operations. We completed second year sampling and
analyses to determine the impact of lactation cycle, local inflammation and
small ruminant lentivirus co-infection on prions in milk, and made progress
adapting a seeded-conversion assay for detecting misfolded prion proteins in
milk. Continued collection and analysis of rectal tissue samples from sheep and
goats of various PrP genotypes will contribute towards improved diagnostic
testing of goats, and the results should be useful for optimization of biopsy
sampling and processing procedures for use with standard diagnostic assays.
Progress on development of blood testing includes using bioassay in sheep and
transgenic mice to determine which blood fractions harbor scrapie prions.
Certain blood fractions could be detected by bioassay even when derived from the
smaller blood sample volumes routinely collected under field conditions.
Accumulation profiles of disease-associated prion protein were compared between
hemal nodes and lymph nodes of sheep and goats to demonstrate the distribution
in the circulatory system. As an alternative to bioassay we have begun
developing additional transgenic cell lines that express species-specific prion
proteins and are permissive to animal-derived as well as culture-adapted scrapie
strains. Other work included determination that only one strain is evident in
the widely studied Stetsonville isolate of transmissible mink encephalopathy and
that TME in mink is a relevant model of extrinsic prion infections of man. The
potential for genetic control of chronic wasting disease in North American
cervids was reviewed and the diagnostic accuracy of rectal biopsy for detecting
chronic wasting disease in captive North American white tailed deer was
determined. We began development of methods to mitigate prion contamination of
soil using a primary cell line of sheep microglia. Further testing of this cell
line found it to be robustly permissive to geographically divergent authentic
sources of scrapie prions. In addition, these cells have been adapted
successfully to a high throughput format. By developing and testing immortalized
ovine and caprine microglial cell lines of relevant prion protein genotypes and
working toward expressing and purifying recombinant ovine and caprine prion
protein that can be utilized in a cell-free misfolding assay we have contributed
to the contingency plan to reduce the use of Tg mice for bioassay. These
advances create the real potential to replace the need for bioassay in sorption
and mitigation studies in soil with more timely and cost efficient in vitro
methods.
4.Accomplishments 1. Detection of scrapie prions in routine blood samples.
The presence of infectious scrapie prions in the blood of some sheep indicates
the possibility of developing a blood-based diagnostic test. However, initial
studies utilized blood volumes much larger than those routinely collected for
diagnostic work. ARS researchers at the Animal Disease Research Unit in Pullman,
Washington, therefore determined if infectious prions could be detected in
volumes of blood typically collected under field conditions. The results
demonstrate that the presence of disease could be determined from these smaller
blood samples after isolating and testing the specific cells known to harbor
scrapie prions. The ability to utilize blood sample volumes routinely collected
under field conditions is an important step toward developing a sensitive
blood-based test for scrapie disease.
2. A live-animal test to monitor chronic wasting disease in free-ranging
elk herds. Keeping track of chronic wasting disease – a fatal prion disease
affecting the brain of deer and elk – is important to managing North America
herds. In some areas, the presence of disease in free-ranging herds can be
monitored by testing tissues collected from hunter-harvested animals, but such
surveillance is generally not possible in herds managed within national parks.
Thus a live-animal test was needed. In this project, ARS researchers at the
Animal Disease Research Unit in Pullman, Washington, teamed up with colleagues
in the National Park Service and at Colorado State University to determine if
biopsy collection of small amounts of tissue from the rectal mucosa could be
used to fill this surveillance gap in free-ranging elk. Despite a limited
ability to detect disease during the early stage of infection in individual elk,
the live-animal test did prove useful in estimating the minimum prevalence of
disease in the herd. Thus, the live animal testing procedure should be a useful
tool for researchers and managers of free-ranging elk herds.
3. The placenta of goats with scrapie is infectious to other goats and
sheep. Scrapie disease has long been known to affect both sheep and goats but
very little is known about the disease specifically in goats. Control of disease
depends on accurate knowledge of the disease process including identifying the
major sources for transmission of disease. In infected sheep, for example, the
placenta shed at lambing is known to be both a major transmission source to
other sheep and goats as well as a source of prions contaminating the
environment. Though this information about the disease in sheep plays a key role
in conducting disease investigations, the role of the placenta shed from goats
with scrapie is not known. In this project, ARS researchers at the Animal
Disease Research Unit in Pullman, Washington, determined that, even though the
pathology associated with scrapie disease in the placenta of goats is
significantly less than that observed in sheep, the goat’s placenta was an
infectious source of scrapie prions to goats and sheep exposed by a natural
route. Thus, like for sheep, the placenta and postpartum period of goats must be
considered transmission risks for other susceptible small ruminants and
environmental contamination.
4. Pathologic prion protein accumulates in a unique blood-filtering organ
of small ruminants. The discovery that the pathologic form of the prion protein
can be detected in in the blood of scrapie infected small ruminants suggests
development of a blood-based test for scrapie may be possible. But little more
is known about the pathologic prion protein in the blood except that it is
present very limited amounts. To learn more its structure and circulation in the
body, ARS researchers at the Animal Disease Research Unit in Pullman,
Washington, studied its accumulation in small blood-filtering organs known as
hemal nodes, an anatomic feature unique to ruminants. The results demonstrate
that the pathologic prion protein in the hemal nodes of sheep and goats is of
similar structure and accumulates at a similar rate and in similar immune cells
as that accumulating in lymph nodes. These results support the conclusion that
the pathologic prion protein circulating in the blood of sheep and goats may be
a suitable target for development of a blood-based diagnostic test.
Review Publications Monello, R., Powers, J.G., Hobbs, N.T., Spraker, T.R.,
O'Rourke, K., Wild, M.A. 2013. Efficacy of antemortem rectal biopsies to
diagnose and estimate prevalence of chronic wasting disease in free-ranging elk
(Cervus elaphus nelsoni). Journal of Wildlife Diseases. DOI:
10.7589/WD.2011-12-362.
Dassanayake, R.P., Truscott, T.C., Özyigit, Ö.M., Zhuang, D., Schneider,
D.A., O'Rourke, K.I. 2013. Accumulation of PrP-Sc in hemal nodes of naturally
and experimentally scrapie-infected sheep. BioMed Central (BMC) Veterinary
Research. doi:10.1186/1746-6148-9-82.
Schneider, D.A., Harrington, R., Zhuang, D., Yan, H., Truscott, T.C.,
Dassanayake, R., Orourke, K.I. 2012. Disease-associated prion protein in neural
and lymphoid tissues of mink (Mustela vison) inoculated with transmissible mink
encephalopathy. Journal of Comparative Pathology. 147:508-521.
Thomsen, B.V., Schneider, D.A., O'Rourke, K., Gidlewski, T., Mclane, J.,
Allen, R.W., Mcisaac, A.A., Mitchell, G.B., Keane, D.P., Spraker, T.,
Balachandran, A. 2012. Diagnostic accuracy of rectal mucosa biopsy testing for
chronic wasting disease within white-tailed deer (Odocoileus virginianus) herds
in North America:Effects of age,sex,polymorphism at PRNP codon 96,and disease
progression. J Vet Diagn Invest. 24(5):878-87.
Stanton, J.B., Schneider, D.A., Dinkel, K.D., Balmer, B.F., Baszler, T.V.,
Mathison, B.A., Boykin, D.W. 2012. Discovery of a novel, monocationic,
small-molecule inhibitor of scrapie prion accumulation in cultured sheep
microglia and rov cells PLoS one. Antimicrobial Agents and Chemotherapy. PloSone
7(11):e51173.
Research Project: Transmission, Differentiation, and Pathobiology of
Transmissible Spongiform Encephalopathies 2013 Annual Report
1a.Objectives (from AD-416): 1. Investigate the pathobiology of atypical
transmissible spongiform encephalopathies (TSEs) in natural hosts. A.
Investigate the pathobiology of atypical scrapie. B. Investigate the
pathobiology of atypical bovine spongiform encephalopathy (BSE). 2. Investigate
the horizontal transmission of TSEs. A. Assess the horizontal transmission of
sheep scrapie in the absence of lambing. B. Determine routes of transmission in
chronic wasting disease (CWD) infected premises. C. Assess oral transmission of
CWD in reindeer. 3. Investigate determinants of CWD persistence. A. Determine
CWD host range using natural routes of transmission. B. Investigate the
pathobiology of CWD.
1b.Approach (from AD-416): The studies will focus on three animal
transmissible spongiform encephalopathy (TSE) agents found in the United States:
bovine spongiform encephalopathy (BSE); scrapie of sheep and goats; and chronic
wasting disease (CWD) of deer, elk, and moose. The research will address sites
of accumulation, routes of infection, environmental persistence, and ante mortem
diagnostics with an emphasis on controlled conditions and natural routes of
infection. Techniques used will include clinical exams, histopathology,
immunohistochemistry and biochemical analysis of proteins. The enhanced
knowledge gained from this work will help mitigate the potential for
unrecognized epidemic expansions of these diseases in populations of animals
that could either directly or indirectly affect food animals.
3.Progress Report: Research efforts directed toward meeting objective 1 of
our project plan, Investigate the pathobiology of atypical TSEs in natural
hosts, include work in previous years starting with the inoculation of animals
for studies designed to address the pathobiology of atypical scrapie, atypical
bovine spongiform encephalopathy (BSE), as well as a genetic version of BSE.
Animals inoculated with atypical scrapie have not yet developed disease.
Atypical BSE animals have developed disease and evaluation of the samples is
currently underway. Animals inoculated with a genetic version of BSE have
developed disease and the manuscript has been published (2012). In addition, we
have investigated the possibility that atypical scrapie was present earlier than
previously detected in the national flock by analyzing archived field isolates
using methods that were unavailable at the time of original diagnosis. Sample
quality was sufficiently degraded that modern methods were not suitable for
evaluation. In research pertaining to objective 2, Investigate the horizontal
transmission of TSEs, we have initiated a study to determine if cohousing
non-lambing scrapie inoculated sheep is sufficient to transmit scrapie to
neonatal lambs. At this time, scrapie inoculated sheep are being co-housed with
pregnant ewes.
4.Accomplishments 1. Partial effectiveness of a common cleaning product to
disinfect prions. Diseases such as mad cow disease, scrapie, and chronic wasting
disease of animals and Creutzfeldt-Jakob disease in humans are collectively
known as transmissible spongiform encephalopathies. They are caused by an
infectious protein, rather than a virus or bacterium, which is notoriously
difficult to inactivate. Decontaminating surfaces or instruments is essential to
prevent potential spread of these diseases. ARS researchers in Ames, IA tested
whether sodium percarbonate, a chemical similar to hydrogen peroxide and
contained in such products as OxiClean, in combination with a detergent (sodium
dodecyl sulfate) could effectively disinfect surfaces contaminated with prions.
The treatment rendered the prions sensitive to degradation with
protein-destroying enzymes, but only limited reduction in infectivity was
observed. Thus, this treatment alone is not sufficient to inactivate prions, but
could be used in combination with other treatments to decontaminate prion
contaminated areas.
2. Determined the likelihood of transmission of chronic wasting disease
(CWD) to cattle. CWD is a prion disease, similar to mad cow disease in cattle,
which naturally occurs in cervid animals, such as deer and elk, in the United
States. Prion diseases are caused by an infectious protein, rather than a virus
or bacterium. The prion that causes CWD is contagious through animal contact and
through the environment, so cattle could be exposed to the agent of CWD through
contact with infected farmed or free-ranging cervids, or exposure to
contaminated premises. The purpose of this study by ARS researchers in Ames, IA,
was to determine whether cattle could become infected with CWD if it was
inoculated directly into the brain, a much more rigorous route of infection.
Only 2 of the 14 cattle inoculated this way developed disease. Additional
studies are required to assess the potential for cattle to develop CWD through a
more natural route of exposure, but a low rate of transmission following
inoculation into the brain suggests a low risk of transmission through contact
with infected animals or a contaminated environment. However, if this ever
occurred it was also determined that currently used diagnostic techniques would
detect and differentiate CWD from other prion diseases in cattle, such as mad
cow disease.
3. Produced a genetically unique calf for studies of inherited bovine
spongiform encephalopathy (BSE). Diseases such as BSE in cattle, more commonly
known as mad cow disease, and Creutzfeldt-Jakob disease (CJD) in humans are
caused by an infectious protein called a prion, rather than a virus or
bacterium. A usually non-harmful form of this prion protein is normally produced
in animals and humans, however, animals and humans born with a certain genetic
form of this protein are more susceptible to developing an inherited form of
these diseases. Proteins are made up of subunits called amino acids, and these
amino acids are encoded by the DNA that makes up genes. In humans, when the
amino acid lysine is encoded by the DNA in a certain position of the prion gene,
instead of the commonly occurring amino acid glutamate, a genetic form of CJD
can occur. Recently, occurrence of this same amino acid in a cow was associated
with a case of BSE. Before this case of BSE, the lysine for glutamate amino acid
substitution had not been identified in cattle. Researchers at ARS, National
Animal Disease Center, Ames, Iowa, in collaboration with Iowa State University
were able to produce a calf with this lysine for glutamate amino acid
substitution. This animal and its progeny will be used to determine if a genetic
form of BSE occurs and for further study of BSE in cattle. It is important to
distinguish whether cases of BSE that arise in the United States are due to an
infectious or genetic cause.
Review Publications Greenlee, J.J., Nicholson, E.M., Smith, J.D., Kunkle,
R.A., Hamir, A.N. 2012. Susceptibility of cattle to the agent of chronic wasting
disease from elk after intracranial inoculation. Journal of Veterinary
Diagnostic Investigation. 24(6):1087-1093.
Lennon, C.W., Ross, W., Martin-Tumasz, S., Toulokhonov, I., Vrentas, C.E.,
Rutherford, S.T., Lee, J.H., Butcher, S.E., Gourse, R.L. 2012. Direct
interactions between the coiled-coil tip of DksA and the trigger loop of RNA
polymerase mediate transcriptional regulation. Genes and Development.
26(23):2634-2646.
Di Bari, M.A., Nonno, R., Castilla, J., D'Agostino, C., Pirisinu, L.,
Riccardi, G., Conte, M., Richt, J., Kunkle, R., Langeveld, J., Vaccari, G.,
Agrimi, U. 2013. Chronic wasting disease in bank voles: characterisation of the
shortest incubation time model for prion diseases. PLoS Pathogens.
9(3):e1003219.
Smith, J.D., Nicholson, E.M., Foster, G.H., Greenlee, J.J. 2013. Exposure
of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl
sulfate renders PrPSc protease sensitive but does not eliminate infectivity.
BioMed Central (BMC) Veterinary Research. 9:8.
Bose, S., Schonenbrucher, H., Richt, J.A., Casey, T., Rasmussen, M.A.,
Kehrli, Jr., M.E., Petrich, J.W. 2013. Fluorescence spectroscopy of the retina
from scrapie-infected mice. Photochemistry and Photobiology. 89(4):864-868.
Available: http://dx.doi.org/10.1111/php.12056.
Comoy, E.E., Mikol, J., Ruchoux, M., Durand, V., Luccantoni-Freire, S.,
Dehen, C., Correia, E., Casalone, C., Richt, J.A., Greenlee, J.J., Torres, J.M.,
Brown, P., Deslys, J. 2013. Evaluation of the zoonotic potential of
transmissible mink encephalopathy. Pathogens. 2:(3)520-532.
Franz, M., Eiden, M., Balkema-Buschmann, A., Greenlee, J., Schatzl, H.,
Fast, C., Richt, J., Hildebrandt, J.P., Groschup, M.H. 2012. Detection of
PrP(Sc) in peripheral tissues of clinically affected cattle after oral challenge
with bovine spongiform encephalopathy. Journal of General Virology.
93(Pt12):2740-2748.
Smith, J.D., Nicholson, E.M., Greenlee, J.J. 2013. Evaluation of a
combinatorial approach to prion inactivation using an oxidizing agent, SDS, and
proteinase K. BioMed Central (BMC) Veterinary Research. 9:151.
Vrentas, C.E., Greenlee, J.J., Baron, T., Caramelli, M., Czub, S.,
Nicholson, E.M. 2013. Stability properties of PrPSc from cattle with
experimental transmissible spongiform encephalopathies: use of a rapid whole
homogenate, protease-free assay. BMC Veterinary Research. 9:167.
Research Project: DETECTION OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY
AGENTS IN LIVESTOCK, WILDLIFE, AGRICULTURAL PRODUCTS, AND THE ENVIRONMENT 2013
Annual Report
1a.Objectives (from AD-416): We will develop highly sensitive diagnostic
tests to detect transmissible spongiform encephalopathy (TSE) in livestock and
wildlife animal species prior to the onset of clinical disease. We will also
develop tests to confirm the presence or absence of TSE disease agents in
ingredients of animal origin and decontaminated environments.
1b.Approach (from AD-416): The threat of BSE continues to affect export
economics for US meat. Meanwhile scrapie continues to influence sheep profits
and herd biosecurity, and CWD is spreading throughout North America. Thus U.S.
animal industry stakeholders have identified detection of the TSE infectious
agent (prions) as a priority biosecurity research issue essential for prevention
of TSE diseases. We will build on our previous successes using mass spectrometry
(MS) for high-sensitivity and specificity in detection of PrPsc as a marker for
TSE infectivity in blood using a hamster scrapie model. We will also develop a
novel PrP-null mouse strain and related myeloma cell culture system for
production of monoclonal antibodies (MAb), which may be specific for PrPsc. We
will then choose MS or MAb and validate our novel diagnostic for preclinical
diagnosis of scrapie in sheep blood. Whereas MS and MAb methods rely on
dissolved samples, contamination of agricultural products and environmental
surfaces is associated with solid samples. So we will produce a cell culture
based assay for TSE infectivity that is surface-adsorbed. After using the
relatively convenient hamster model for early development, we will validate this
technology for detection of scrapie in sheep brain on meat-and-bone meal and
stainless steel. All work with infectious material will take place within our
APHIS-approved BL2 biocontainment facilities labs at the Western Regional
Research Center (WRRC), while mass spectrometry will be performed on
non-infectious material under BL1 containment. Replacing 5325-32000-007-00D
(3/19/2008).
3.Progress Report: This is the final report for this project which
terminated in April of 2013 and was replaced with 5325-32000-009-00D,
"Innumodiagnostics to Detect Prions and Other Important Animal Pathogens".
Substantial results were realized over the project period. New analytical
techniques and materials were developed for detection of infectious prions with
improved results compared to conventional methods. A prion gene knockout mouse
was developed and immunized with purified infectious prions resulting in the
generation of sensitive anti-prion monoclonal antibodies. The impact of this
research was the production of distinct anti-prion monoclonal antibodies that
bind novel prion epitopes for use in the development of commercial immunoassays.
The binding properties of these monoclonal antibodies were extensively
characterized and their utility in a variety of immunoassay formats were
demonstrated. A patent has been filed for this technology and three of these
antibodies have been transferred to academic and government partners including
the National Veterinary Services Laboratory (NVSL). License negotiations are
currently ongoing with a commercial partner. The slow accumulation of prions and
limited concentration in target tissues confounds detection. Sample preparative
methods were defined that significantly enrich prions in tissue extract using
isolation with lipid raft. The resulting increase in prion sample concentration
results in improved immunoassay detection. The impact of this research is a
method to detect prions from infected asymptomatic animal tissue. A patent has
been filed and papers published on this methodology. Enzyme-linked immunosorbent
assays (ELSIA) are frequently used for the detection of prions from target
tissues. A chaotropic agent was effectively used in a modified
immunohistochemical assay and an ELISA format to increase the sensitivity of
prion detection. This technical modification resulted in a in a seminal paper
with a broad impact on antigen detection by immunoassay. Infectious prion
strains reflect alternate abnormal tertiary protein structure that stably
propagate and produce distinct disease pathology. Analytical mass spectrometry
has been used to elucidate structural differences in prion protein strains and
quantitate the concentration of prion in a given sample. The impact of this
research has been to further the understanding of prion strain differences and
identify potential anti-prion binding epitopes for selective modification. This
knowledge can be used to build immunoassays capable of discriminating prion
strains. The overall impact of these accomplishments is improved prion
immunoassay technologies for use in ongoing prion research and surveillance
efforts with commercial application.
4.Accomplishments 1. Improved anti-prion monoclonal antibodies for prion
detection. Using purified prions from infected animals, ARS scientist in Albany,
California, have generated anti-prion DRM monoclonal antibodies from immunized
prion knockout mice. This approach effectively overcame the poor immune response
to prion immunization in normal mice and our research efforts defined an
improved method to yield a purified infectious prion immunogen used to generate
high-titer antibody response in a new mouse model. A novel differential
screening methods based on preferential binding to infectious prion antigen was
used in the identification and selection of hybridoma cell lines producing
anti-prion monoclonal antibodies. The properties of these antibodies have been
extensively characterized, their binding epitopes defined and their utility in a
number of immunoassay formats validated. These antibodies have been transferred
to both academic and national laboratories for use in prion immunoassays. A
patent has been filed (US Patent # 13/157,216) and a manuscript has been
published describing these anti-prion monoclonal antibodies. Negotiations with a
major international biotechnology company are in progress to issue a license for
commercialization of this technology.
2. Novel peptide sequences for specific binding to infectious prions. Using
a proteomic peptide array consisting of peptides corresponding to functional
domains of endogenous proteins involved in protein-protein interaction ARS
scientists in Albany, California, have identified specific amino acid sequences
that bind only the infectious form of prion proteins. Using these peptides as
part of a modified immunoassay for the selective capture of infectious prion
protein from animal tissues, ARS scientists have demonstrated the ability to
discriminate prion infected from uninfected samples using conventional
anti-prion antibodies for detection. This approach allows for the direct capture
and detection of infectious prions without the interference of the normal
endogenous prion protein that confounds traditional assay techniques. The
identification of several prion specific binding peptides represents important
components of new peptide-based detection assays and important targets to
facilitate research toward understanding the mechanism of prion conformational
conversion. A patent has been issued (US Patent #12/571,275; Aug 2013) for this
technology.
3. Novel methods to isolate prions in non-diagnostic tissues to improve
detection of scrapie and chronic wasting disease (CWD). ARS scientists in
Albany, California, along with an international consortium of scientists have
developed an efficient means of isolating and detecting prions in non-diagnostic
portions of brain tissue from sheep naturally infected with scrapie and elk
infected with CWD. Conventional methods rely on accumulation of infectious
prions in a brainstem tissue called the obex for diagnosis of disease. Other
areas of the brain tend to have less prion and consequently have been excluded
from sampling and analysis. The small size and lipid rich aspect of the obex has
limited the application of prion detection assays. ARS scientists have devised
new methods to enhance prion detection from brain regions that have been
previously ignored for use in confirmatory diagnostic testing. These results
have been published in the scientific literature and provide alternate
techniques to augment current prion testing protocols.
Review Publications Stanker, L.H., Scotcher, M.C., Lin, A.V., Mcgarvey,
J.A., Prusiner, S., Hnasko, R.M. 2012. Novel epitopes identified by Anti-PrP
monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice
with purified scrapie prions. Hybridoma. 31(5):312-324.
doi:10.1089/hyb.2012.0022.
Vasquez-Fernandez, E., Alonso, J., Pastrana, M.A., Ramos, A., Stitz, L.,
Vidal, E., Dynin, I.A., Petsch, B., Silva, C.J., Requena, J.R. 2012. Structural
organization of mammalian prions as probed by limited proteolysis. PLoS One.
7(11):e50111. doi:10.1371/journal.pone.0050111.
Silva, C.J., Dynin, I.A., Erickson-Beltran, M.L., Requena, J.R.,
Balachandran, A., Onisko, B.C., Hui, C., Carter, J.M. 2013. Using mass
spectrometry to detect prions and oxidized prions in scrapie-infected sheep and
CWD-infected elk. Biochemistry. 52:2139-2147. doi:10.1021/bi3016795.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF
TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Title: Atypical BSE: role of the E211K
prion polymorphism
Author
item Greenlee, Justin
Submitted to: American Veterinary Medical Association Abstract Publication
Type: Abstract Only Publication Acceptance Date: May 1, 2013 Publication Date:
July 19, 2013 Citation: Greenlee, J.J. 2013. Atypical BSE: role of the E211K
prion polymorphism. 2013 American Veterinary Medical Association (AVMA) Annual
Convention, July 19-23, 2013, Chicago, Illinois. Paper No. 13920. p. 45.
Technical Abstract: Prion diseases or transmissible spongiform
encephalopathies (TSEs) are fatal neurodegenerative diseases that naturally
affect several species including human beings. These chronic diseases are
associated with the accumulation of a protease-resistant, disease-associated
isoform of the prion protein (PrPSc) in the central nervous system and other
tissues, depending on the species affected. In humans, TSEs can be acquired
through exposure to infectious material, inherited as germline polymorphisms in
the prion gene (prnp), or occur spontaneously. Bovine spongiform encephalopathy
(BSE) or mad cow disease cases can be subclassified into at least 3 distinct
disease forms with the predominant form known as classical BSE and the others
collectively referred to as atypical BSE. Atypical BSE is further subdivided
into H-type and L-type cases that are distinct from classical BSE and from each
other on the basis of their unique molecular profiles. Both atypical BSE
subtypes are believed to have arisen spontaneously and today researchers are
investigating atypical BSE as a possible origin of classical BSE that was a
feed-borne epidemic primarily affecting cattle in Europe where BSE-contaminated
animal protein sources derived from central nervous system tissues were
previously fed to ruminants. Several hypotheses have been proposed to explain
atypical BSE cases. At the forefront of this discussion is the possibility that
both H-type and L-type BSE may be spontaneous diseases in cattle. Support for
atypical BSE occurring spontaneously is drawn from parallels to sporadic prion
disease in humans, specifically, occurrence in older hosts and a comparable low
incidence rate. Furthermore, atypical BSE occurs as isolated, sporadic cases in
contrast to the clustering of cases observed for feedborne classical BSE.
Recognition of a spontaneous prion disease in cattle, coupled with evidence
indicating atypical BSE can convert to classical BSE upon serial passage in
mice, has broad implications for our understanding of the origins of the
classical form of the disease. In 2006 a critical discovery was made when one
case of H-type BSE was associated with a heritable mutation in the prion protein
gene referred to as E211K. This case was diagnosed in the U.S. and led to the
identification of a new prion protein (prnp) allele, K211, that is associated
with H-type BSE and is heritable. This presentation will present data
demonstrating a rapid onset of disease in an animal with the E211K mutation
following experimental inoculation with H-type BSE from the original E211K
H-type BSE case. Interestingly, disease associated prion protein was widespread
in neural tissues this animal, and antemortem retinal thinning and functional
deficits of the visual system were observed prior to the onset of clinical
disease. The existence of genetic forms of BSE offers new explanations for the
potential origins of BSE.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF
TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Title: Evaluation of the zoonotic
potential of transmissible mink encephalopathy
Authors
item Comoy, Emmanuel - item Mikol, Jacqueline - item Ruchoux,
Marie-Madeleine - item Durand, Valerie - item Luccantoni-Freire, Sophie - item
Dehen, Capucine - item Correia, Evelyne - item Casalone, Cristina - item Richt,
Juergen item Greenlee, Justin item Torres, Juan Maria - item Brown, Paul - item
Deslys, Jean-Philippe -
Submitted to: Pathogens Publication Type: Peer Reviewed Journal Publication
Acceptance Date: July 30, 2013 Publication Date: July 30, 2013 Citation: Comoy,
E.E., Mikol, J., Ruchoux, M., Durand, V., Luccantoni-Freire, S., Dehen, C.,
Correia, E., Casalone, C., Richt, J.A., Greenlee, J.J., Torres, J.M., Brown, P.,
Deslys, J. 2013. Evaluation of the zoonotic potential of transmissible mink
encephalopathy. Pathogens. 2:(3)520-532.
Interpretive Summary: Cases of bovine spongiform encephalopathy (BSE) or
mad cow disease can be subclassified into at least 3 distinct disease forms with
the predominate form known as classical BSE and the others collectively referred
to as atypical BSE. Atypical BSE can be further subdivided into H-type and
L-type cases that are distinct from classical BSE and from each other. Both of
the atypical BSE subtypes are believed to occur spontaneously, whereas classical
BSE is spread through feeding contaminated meat and bone meal to cattle.
Transmissible mink encephalopathy (TME) is another prion disease that transmits
to cattle and show similarities to L-type BSE when subjected to laboratory
testing. The purpose of this study was to use non-human primates (cynomologous
macaque) and transgenic mice expressing the human prion protein to determine if
TME could represent a potential risk to human health. TME from two sources
(cattle and raccoons) was able to infect non-human primates and transgenic mice
after exposure by the intracranial route. This result suggest that humans may be
able to replicate TME prions after an exposure that allows infectious material
access to brain tissue. At this time, it is unknown whether non-human primates
or transgenic mice would be susceptible to TME prions after oral exposure. The
results obtained in these animal models were similar to those obtained for
L-type BSE. Although rare, the existence of TME and that it transmits to cattle,
non-human primates, and transgenic mice suggest that feed bans preventing the
feeding of mammalian tissues to cattle should stay in place and that regular
prion surveillance during the slaughter should remain in place. Parties with
interest in the cattle and beef industries and regulatory officials responsible
for safe feeding practices of cattle will be interested in this work. Technical
Abstract: Successful transmission of Transmissible Mink Encephalopathy (TME) to
cattle supports the bovine hypothesis to the still controversial origin of TME
outbreaks. Human and primate susceptibility to classical Bovine Spongiform
Encephalopathy (c-BSE) and the transmissibility of L-type BSE to macaques assume
a low cattle-to-primate species barrier: we therefore evaluated the zoonotic
potential of cattle-adapted TME. In less than two years, this strain induced in
cynomolgus macaques a neurological disease similar to L-BSE and distinct from
c-BSE. TME derived from another donor species (raccoon) induced a similar
disease with shorter incubation periods. L-BSE and cattle-adapted TME were also
transmissible to transgenic mice expressing human PrP. Interestingly, secondary
transmissions to transgenic mice expressing bovine PrP showed the maintenance of
prion strain features for the three tested bovine prion strains (cattle TME,
c-BSE and L-BSE) regardless of intermediate host. Thus, TME is the third animal
prion strain transmissible to both macaques and humanized transgenic mice,
suggesting zoonotic potentials that should be considered in the risk analysis of
animal prion diseases for human health. Moreover, the similarities between TME
and L-BSE are highly suggestive of a link between those strains, and of the
presence of L-BSE decades prior to its identification in USA and Europe.
TME in mink was documented in the early 1960s. it was first thought that
the TME out break was from scrapie infected sheep, until a investigation was
done on feed practices at these mink facilities, and it was later found that the
mink had been fed 95%+ dead stock downer cows. and later, the Late Richard Marsh
tried to warn the feds of the pending mad cow debacle. they refused to listen.
...
some interesting reading on pages 26 to 33
1979
TME originates from feeding mink, scrapie infected materials...
Evidence That Transmissible Mink Encephalopathy Results from Feeding
Infected Cattle
Over the next 8-10 weeks, approximately 40% of all the adult mink on the
farm died from TME.
snip...
The rancher was a ''dead stock'' feeder using mostly (>95%) downer or
dead dairy cattle...
another nice bit of history on cwd can be found here ;
I know scarpie was first documented around 1947 here in the USA, how long
it was here before that, I don’t know, some say over 250 years.
Saturday, June 25, 2011
Transmissibility of BSE-L and Cattle-Adapted TME Prion Strain to Cynomolgus
Macaque
"BSE-L in North America may have existed for decades"
Sunday, December 15, 2013
FDA PART 589 -- SUBSTANCES PROHIBITED FROM USE IN ANIMAL FOOD OR FEED
VIOLATIONS OFFICIAL ACTION INDICATED OAI UPDATE DECEMBER 2013 UPDATE
Thursday, July 24, 2014
Protocol for further laboratory investigations into the distribution of
infectivity of Atypical BSE SCIENTIFIC REPORT OF EFSA
Discussion: The C, L and H type BSE cases in Canada exhibit molecular
characteristics similar to those described for classical and atypical BSE cases
from Europe and Japan. *** This supports the theory that the importation of BSE
contaminated feedstuff is the source of C-type BSE in Canada. *** It also
suggests a similar cause or source for atypical BSE in these countries. ***
see page 176 of 201 pages...tss
*** Singeltary reply ; Molecular, Biochemical and Genetic Characteristics
of BSE in Canada Singeltary reply ;
Saturday, August 14, 2010
BSE Case Associated with Prion Protein Gene Mutation (g-h-BSEalabama) and
VPSPr PRIONPATHY
(see mad cow feed in COMMERCE IN ALABAMA...TSS)
PRION 2014 CONFERENCE
CHRONIC WASTING DISEASE CWD
A FEW FINDINGS ;
Conclusions. To our knowledge, this is the first established experimental
model of CWD in TgSB3985. We found evidence for co-existence or divergence of
two CWD strains adapted to Tga20 mice and their replication in TgSB3985 mice.
Finally, we observed phenotypic differences between cervid-derived CWD and
CWD/Tg20 strains upon propagation in TgSB3985 mice. Further studies are underway
to characterize these strains.
We conclude that TSE infectivity is likely to survive burial for long time
periods with minimal loss of infectivity and limited movement from the original
burial site. However PMCA results have shown that there is the potential for
rainwater to elute TSE related material from soil which could lead to the
contamination of a wider area. These experiments reinforce the importance of
risk assessment when disposing of TSE risk materials.
The results show that even highly diluted PrPSc can bind efficiently to
polypropylene, stainless steel, glass, wood and stone and propagate the
conversion of normal prion protein. For in vivo experiments, hamsters were ic
injected with implants incubated in 1% 263K-infected brain homogenate. Hamsters,
inoculated with 263K-contaminated implants of all groups, developed typical
signs of prion disease, whereas control animals inoculated with non-contaminated
materials did not.
Our data establish that meadow voles are permissive to CWD via peripheral
exposure route, suggesting they could serve as an environmental reservoir for
CWD. Additionally, our data are consistent with the hypothesis that at least two
strains of CWD circulate in naturally-infected cervid populations and provide
evidence that meadow voles are a useful tool for CWD strain typing.
Conclusion. CWD prions are shed in saliva and urine of infected deer as
early as 3 months post infection and throughout the subsequent >1.5 year
course of infection. In current work we are examining the relationship of
prionemia to excretion and the impact of excreted prion binding to surfaces and
particulates in the environment.
Conclusion. CWD prions (as inferred by prion seeding activity by RT-QuIC)
are shed in urine of infected deer as early as 6 months post inoculation and
throughout the subsequent disease course. Further studies are in progress
refining the real-time urinary prion assay sensitivity and we are examining more
closely the excretion time frame, magnitude, and sample variables in
relationship to inoculation route and prionemia in naturally and experimentally
CWD-infected cervids.
Conclusions. Our results suggested that the odds of infection for CWD is
likely controlled by areas that congregate deer thus increasing direct
transmission (deer-to-deer interactions) or indirect transmission
(deer-to-environment) by sharing or depositing infectious prion proteins in
these preferred habitats. Epidemiology of CWD in the eastern U.S. is likely
controlled by separate factors than found in the Midwestern and endemic areas
for CWD and can assist in performing more efficient surveillance efforts for the
region.
Conclusions. During the pre-symptomatic stage of CWD infection and
throughout the course of disease deer may be shedding multiple LD50 doses per
day in their saliva. CWD prion shedding through saliva and excreta may account
for the unprecedented spread of this prion disease in nature.
see full text and more ;
Monday, June 23, 2014
*** PRION 2014 CHRONIC WASTING DISEASE CWD
Thursday, July 03, 2014
*** How Chronic Wasting Disease is affecting deer population and what’s the
risk to humans and pets?
Tuesday, July 01, 2014
*** CHRONIC WASTING DISEASE CWD TSE PRION DISEASE, GAME FARMS, AND
POTENTIAL RISK FACTORS THERE FROM
***P.170: Potential detection of oral transmission of H type atypical BSE
in cattle using in vitro conversion
Sandor Dudas, John G Gray, Renee Clark, and Stefanie Czub Canadian Food
Inspection Agency; Lethbridge, AB Canada
Keywords: Atypical BSE, oral transmission, RT-QuIC
The detection of bovine spongiform encephalopathy (BSE) has had a
significant negative impact on the cattle industry worldwide. In response,
governments took actions to prevent transmission and additional threats to
animal health and food safety. While these measures seem to be effective for
controlling classical BSE, the more recently discovered atypical BSE has
presented a new challenge. To generate data for risk assessment and control
measures, we have challenged cattle orally with atypical BSE to determine
transmissibility and mis-folded prion (PrPSc) tissue distribution. Upon
presentation of clinical symptoms, animals were euthanized and tested for
characteristic histopathological changes as well as PrPSc deposition.
The H-type challenged animal displayed vacuolation exclusively in rostral
brain areas but the L-type challenged animal showed no evidence thereof. To our
surprise, neither of the animals euthanized, which were displaying clinical
signs indicative of BSE, showed conclusive mis-folded prion accumulation in the
brain or gut using standard molecular or immunohistochemical assays. To confirm
presence or absence of prion infectivity, we employed an optimized real-time
quaking induced conversion (RT-QuIC) assay developed at the Rocky Mountain
Laboratory, Hamilton, USA.
Detection of PrPSc was unsuccessful for brain samples tests from the orally
inoculated L type animal using the RT-QuIC. It is possible that these negative
results were related to the tissue sampling locations or that type specific
optimization is needed to detect PrPSc in this animal. We were however able to
consistently detect the presence of mis-folded prions in the brain of the H-type
inoculated animal. Considering the negative and inconclusive results with other
PrPSc detection methods, positive results using the optimized RT-QuIC suggests
the method is extremely sensitive for H-type BSE detection. This may be evidence
of the first successful oral transmission of H type atypical BSE in cattle and
additional investigation of samples from these animals are ongoing.
P.169: PrPSc distribution in brain areas of a natural German H-type BSE
case
Anne Balkema-Buschmann, Grit Priemer, Markus Keller, and Martin H Groschup
Friedrich Loeffler Institut, Institute for Novel and Emerging Infectious
Diseases; Greifswald, Insel Riems, Germany
Keywords: BSE H-type, brain, muscle
Ten years after the initial description of atypical BSE cases of the H-type
and L-type, the distribution of PrPSc in different brain areas and peripheral
tissues of natural cases of these BSE forms is still not fully understood.
Intracerebral challenge experiments have been performed with both atypical BSE
forms in cattle, and the distribution of the abnormal prion protein and
infectivity has been analysed in a variety of tissues, confirming the general
restriction to the central nervous system as it was already generally
acknowledged for classical BSE, but showing a slightly earlier and stronger
involvement of the peripheral nervous system and the skeletal muscle.
www.landesbioscience.com Prion 105
However, data from cattle orally challenged with atypical BSE, which might
mimic the natural situation, are not yet available. Unfortunately, for most
natural cases of atypical BSE, only the obex region is available for further
analysis. The PrPSc distribution in the brains of natural L-type BSE cases in
Italy has been described in some detail, but comparably few such data are yet
available for natural H-type cases. Here we describe the analysis of different
brain areas and muscle samples of a natural H-type BSE case diagnosed in Germany
in 2014, and compare these data with those obtained from the respective samples
collected from cattle challenged intracerebrally with H-type BSE.
P.159: Transgenic mice overexpressing rabbit prion protein are susceptible
to BSE, BASE and scrapie prion strains but resistant to CWD and atypical
scrapie
Natalia Fernández-Borges,1 Enric Vidal,2 Belén Pintado,4 Hasier Eraña,1
Montserrat Ordóñez,3 Mercedes Márquez,5 Francesca Chianini,6 Dolors Fondevila,5
Manuel A Sánchez-Martín,7 Olivier Andréoletti,8 Mark P Dagleish,6 Martí
Pumarola,5 and Joaquín Castilla1,3 1CIC bioGUNE; Parque tecnológico de Bizkaia;
Derio; Bizkaia, Spain; 2Centre de Recerca en Sanitat Animal (CReSA); UAB-IR TA,
Campus de la Universitat Autònoma de Barcelona; Bellaterra; Barcelona,
Catalonia, Spain; 3IKERBASQUE; Basque Foundation for Science; Bilbao, Bizkaia,
Spain; 4Centro Nacional de Biotecnología (CNB), Campus de Cantoblanco;
Cantoblanco; Madrid, Spain; 5Department of Animal Medicine and Surgery;
Veterinary faculty; Universitat Autònoma de Barcelona (UAB); Bellaterra
(Cerdanyola del Vallès); Barcelona, Catalonia, Spain; 6Moredun Research
Institute; Bush Loan, Penicuik, Scotland, UK; 7Unidad de Generación de OMGs.
S.E.A. Department of Medicine; University of Salamanca; Salamanca, Spain; 8Ecole
Nationale du Veterinaire; Service de Pathologie du Bétail; Toulouse,
France
Interspecies transmission of prions is a well established phenomenon, both
experimentally and in field conditions. Upon passage through new hosts prion
strains have proven their capacity to change their properties. It is, in fact, a
source of strain diversity which needs to be considered when assessing the
potential risks associated with consumption of prion contaminated protein
sources.
Rabbits were considered for decades a prion resistant species until proven
recently otherwise. To determine the extent of rabbit susceptibility to prions
and to assess their effects on the passage of different prion strains through
this species, a transgenic mouse model overexpressing rabbit PrPC was developed
(TgRab). Intracerebral challenges with prion strains originating from a variety
of species including field isolates (SSBP1 scrapie, Nor98-like scrapie, BSE,
BASE and CWD), experimental murine strains (ME7 and RML), experimentally
obtained strains (sheepBSE) and strains obtained by in vitro crossing of the
species barrier using saPMCA (BSE-RabPrPres, SSBP1-RabPrPres and CWD-RabPrPres)
have been performed.
Interestingly, on first passage, TgRab were susceptible to the majority of
prions tested with the exception of SSBP1 scrapie, CWD and Nor98 scrapie.
Furthermore TgRab were capable of propagating strain-specific features such as
differences in incubation periods, brain lesion and PrPd deposition profiles and
PK resistant western blotting band patterns. Our results confirm previous
studies shattering the myth that rabbits are resistant to prion infection and
this should be taken into account when choosing protein sources to feed rabbits.
P.168: Evolution of the biological properties of L-BSE after passage in
sheep with susceptible and resistant PrP genotypes
Michele A Di Bari, Umberto Agrimi, Claudia D’Agostino, Geraldina Riccardi,
Stefano Marcon, Elena Esposito, Paolo Frassanito, Flavio Torriani, Shimon
Simson, and Romolo Nonno Istituto Superiore di Sanità (ISS) Department of
Veterinary Public Health and Food Safety; Rome, Italy
Background. Cattle L-BSE was efficiently transmitted to sheep with
susceptible (QQ171) and resistant (QR171) PrP genotypes. 1 Notably, the PrPSc
signature of L-BSE was preserved in QQ171 sheep but not in QR171 sheep.2
Notwithstanding, bioassay in transgenic mice expressing bovine or ovine (ARQ)
PrPC showed that L-BSE strain was preserved in both, QQ171 and QR171
sheep-passaged L-BSE.3
Here we studied the biological properties of sheep-passaged L-BSE by
bioassay in bank voles and transgenic mice expressing the ovine VRQ PrP (tg338),
both characterized by a comparatively low susceptibility to cattle L-BSE.
Material and Methods. Voles and tg338 mice were intracerebrally inoculated
with cattle L-BSE and sheep-passaged (QQ171 and QR171) L-BSE isolates. Survival
time, lesion profiles, Pet-blot and WB analysis were used for strain typing.
Results. Cattle L-BSE transmitted quite inefficiently to tg338 mice, with
survival time >400 days post-infection (d.p.i.), while sheep-passaged inocula
were much more efficient and all gave terminal disease by ~140 d.p.i. However,
after sub-passage all inocula converged to a survival time of ~145 d.p.i.. and
showed overlapping pathological phenotypes.
In voles, cattle L-BSE transmitted with very long survival times (~800
d.p.i.) and was accompanied by an upward shift of the PrPSc type. Again, all
sheep-passaged L-BSE isolates transmitted much more efficiently, with similar
survival times of ~360 d.p.i.. Upon second passage, three different strains were
isolated in vole, characterized by distinct pathological phenotypes. This
divergence is epitomized by the different survival times of vole-adapted L-BSE
strains, which were ~400 d.p.i. for cattle L-BSE, ~130 d.p.i. for QQ171-passaged
L-BSE and ~225 d.p.i. for QR171-passaged L-BSE.
Conclusions. These findings, along with previously published data,3 show
that the original L-BSE strain was recovered after passage in sheep when
bioassay was performed in animal models expressing bovine or ovine PrPC. In
contrast, strain changes were observed in both, QQ171- and QR171-passaged L-BSE
by bioassay in vole, a species with divergent PrP sequence compared to
ruminants. Importantly, QQ171- and QR171-passaged L-BSE were characterised by
different PrPSc types and, accordingly, showed different biological properties
when transmitted to voles, but not when transmitted to other animal models.
Overall, our work support the hypothesis that prion isolates are likely
composed of multiple prion components, emphasizes the role of host PrP
polymorphisms on strain selection and mutation, and highlights the risk for new
potentially zoonotic strains that could emerge from prion evolution in animal
reservoirs.
P.172: BSE exposure risk from bovine intestine and mesentery
Fulvio Barizzone,1 Herbert Budka,2 Christine Fast,3 John N Griffin,4
Giuseppe Ru,5 Pietro Stella1 and Olivier Andréoletti6 1European Food Safety
Authority; Parma, Italy; 2Institute of Neuropathology; University Hospital
Zurich; Zurich, Switzerland; 3Friedrich-Loeffler-Institut; Institute of Novel
and Emerging Infectious Diseases; Isle of Riems, Germany; 4Department of
Agriculture, Food and the Marine; Backweston, Celbridge, Co. Kildare, Ireland;
5Istituto Zooprofilattico Sperimentale del Piemonte; Liguria e Valle d’Aosta;
Biostatistics Epidemiology and Analysis of Risk (BEAR) unit; Turin, Italy; 6UMR
Interactions Hôtes Agents Pathogènes; Ecole Nationale Vétérinaire INR A; ENVT;
Toulouse, France
Keywords: Bovine Spongiform Encephalopathy (BSE), cattle, intestine,
mesentery, specified risk material (SRM), quantitative risk assessment
(QRA)
Bovine intestines and mesenteries in the European Union (EU) are considered
among the tissues potentially containing the highest level of BSE infectivity
and have to be removed from the food and feed chain. A quantitative assessment
of the BSE infectious load potentially entering the food and feed chain yearly
in the European Union (EU) was developed. The evolution of the BSE infectious
titre and of the weight of the structures accumulating infectivity was
considered. The number of BSE infected cattle entering undetected in the food
and feed chain yearly was estimated. A model (TSEi) was developed to estimates
the evolution of the BSE infectious load in animals and the total yearly
infectious load that could enter the food and feed chain. In a BSE infected
bovine, the distribution of infectivity in intestines and mesentery varies with
the age. Up to 36 months of age the infectivity is mainly associated (on average
more than 90%) with the last 4 metres of small intestine and the caecum, over 36
and under 60 months of age, there is an inter-individual variability, from 60
months of age the infectivity is mainly associated (on average more than 90%)
with the mesenteric nerves and the celiac and mesenteric ganglion complex. The
total amount of infectivity peaks, about 15 BoID50, in animals younger than 18
months, it declines to 8-9 BoID50 (24–48 months of age) and it drops to 0.7
BoID50 in animals older than 60 months. The ileocaecal plate is the most
infectious part of the intestine and it can be used to estimate the potential
maximum level of exposure for an individual consumer.
In the EU, between 2007 and 2012, the yearly amount of BSE infectivity
associated with intestine and mesentery from animals entering the food and feed
chain was reduced by a factor of 10 (from about 23,000 to about 2,000 BoID50).
However, the maximum level of exposure to the BSE agent from intestine
remained stable (on average about 1.5-1.6 BoID50 per meter).
In case of re-emergence of BSE in the EU there would be an increase of the
potential maximum level of exposure to BSE from intestine. According to the TSEi
model the removal of the last four metres of the small intestine and of the
caecum from the food and feed chain would result in a major reduction of the BSE
exposure risk associated with intestine and mesentery in cattle.
P.131: Transmission of sheep-bovine spongiform encephalopathy in pigs
Carlos Hedman,1 Belén Marín,1 Fabian Corbière,3 Hicham Filali,1 Francisco
Vázquez, José Luis Pitarch,1 William Jirón,1 Rodrigo S Hernandez,1 Bernardino
Moreno,1 Martí Pumarola,2 Olivier Andréoletti,3 Juan José Badiola,1 and Rosa
Bolea1 1University of Zaragoza; Zaragoza, Spain; 2University of Barcelona;
Barcelona, Spain; 3Institut National de la Recherche (INR A); Toulouse,
France
Introduction. The transmissible spongiform encephalopathies (TSE) don´t
occur in swine in natural conditions. However, the bovine spongiform
encephalopathy (BSE) agent, inoculated by 3 simultaneous routes in pigs, is able
to reproduce a neurological disease in these animals. On the other hand, the BSE
agent after passage in sheep under experimental conditions (sheep- BSE) exhibits
altered pathobiologic properties. This new agent is able to cross the cattle-pig
transmission barrier more efficiently than BSE. The potential propagation of TSE
in animals from the human food chain, including pigs, needs to be assessed
regarding the risk for human infection by animals other than TSE-infected
ruminants. The aim of this work was to determine the susceptibility of pigs to
the Sheep-BSE agent and describe the pathological findings and PrPSc deposition
in different tissues.
Material and Methods. Seven minipigs were challenged intracerebrally with
sheep-BSE agent. Clinical observation and postmortem histopathology,
immunohistochemistry (antibody 2G11) and Western blotting were performed on
central nervous system (CNS), peripheral nervous system (PNS) and other
tissues.
Results. One pig was culled in an early incubation stage, and remaining six
were culled at the presence of clinical sings. Pigs developed a clinical disease
with locomotor disorders in an average time of 23 months post inoculation,
showing clinical findings in most of them earlier than those described in the
BSE in pigs experimental infection. TSE wasn´t confirmed in the preclinical pig.
In clinical pigs, the entire cerebral cortex showed severe neuropil vacuolation,
extensive and severe vacuolar changes affecting the thalamus, hippocampus and
cerebellum. PrPSc was found in CNS of all clinical pigs (6/6). Intracellular
(intraneuronal and intraglial) and neuropil-associated PrPSc deposition was
consistently observed in the brainstem, thalamus, and deeper layers of the
cerebral cortex. Also, PrPSc was observed in PNS, mainly in the myenteric plexus
and also in nerves belonging to the skeleton muscle. Moreover, the glycosylation
profile showed a 3 band pattern with a predominant monoglycosylated band in
positive pig samples.
This features concern on the potential risk of utilization of meat and
bound meal of small ruminants in feeding pigs.
P.177: Elements modulating the prion species barrier and its passage
consequences
Juan-Carlos Espinosa,1 Patricia Aguilar-Calvo,1 Ana Villa-Diaz,1 Olivier
Andréoletti,2 and Juan María Torres1 1Centro de Investigación en Sanidad Animal
(CISA-INI A); Valdeolmos, Madrid, Spain; 2UMR INR A-ENVT 1225; Interactions Hôte
Agent Pathogène; École Nationale Vétérinaire de Toulouse; Toulouse, France
The phenotypic features of Transmissible Spongiform Encephalopathy (TSE)
strains may be modified during passage across a species barrier. In this study
we investigated the biochemical and biological characteristics of Bovine
Spongiform Encephalopathy (BSE) infectious agent after transmission in both
natural host species (cattle, sheep, pigs, and mice) and in transgenic mice
overexpressing the corresponding cellular prion protein (PrPC) in comparison
with other non-BSE related prions from the same species. After these passages,
most characteristics of the BSE agent remained unchanged. BSE-derived agents
only showed slight modifications in the biochemical properties of the
accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation
into transgenic mice expressing bovine-PrPC. Transmission experiments in
transgenic mice expressing bovine, porcine or human-PrP revealed that all
BSE-derived agents were transmitted with no or a weak transmission barrier. In
contrast, a high species barrier was observed for the non-BSE related prions
that harboured an identical PrP amino acid sequence such as sheep-scrapie, mouse
RML or human sCJD isolates, supporting the theory that the prion transmission
barrier is modulated by strain properties (presumably conformation-dependent)
rather than by PrP amino acid sequence differences between host and donor.
As identical results were observed with prions propagated either in natural
hosts or in transgenic mouse models, we postulate that the species barrier and
its passage consequences are uniquely governed by the host PrPC sequence and not
influenced by the PrPC expression level or genetic factors other than the PrPC
amino acid sequence. All these findings unequivocally demonstrate that the
species barrier and its passage consequences are uniquely driven by the PrPC
sequence, and not by other host genetic factors, demonstrating the validity of
transgenic PrP animals as models for studies of the species barrier.
The results presented herein reinforce the idea that the BSE agent is
highly promiscuous, infecting other species, maintaining its properties in the
new species, and even increasing its capabilities to jump to other species
including humans. These data are essential for the development of an accurate
risk assessment for BSE.
SNIP...SEE FULL TEXT ;
Monday, June 23, 2014
*** PRION 2014 TYPICAL AND ATYPICAL BSE AND CJD REPORT UPDATES
Transmissible Spongiform Encephalopathy TSE Prion Disease North America
2014
Transmissible Spongiform Encephalopathy TSE Prion Disease have now been
discovered in a wide verity of species across North America. typical C-BSE,
atypical L-type BASE BSE, atypical H-type BSE, atypical H-G BSE, of the bovine,
typical and atypical Scrapie strains, in sheep and goats, with atypical Nor-98
Scrapie spreading coast to coast in about 5 years. Chronic Wasting Disease CWD
in cervid is slowly spreading without any stopping it in Canada and the USA and
now has mutated into many different strains. Transmissible Mink Encephalopathy
TME outbreaks. These Transmissible Spongiform Encephalopathy TSE Prion Disease
have been silently mutating and spreading in different species in North America
for decades.
The USDA, FDA, et al have assured us of a robust Triple BSE TSE prion
Firewall, of which we now know without a doubt, that it was nothing but ink on
paper. Since the 1997 mad cow feed ban in the USA, literally tons and tons of
banned mad cow feed has been put out into commerce, never to return, as late as
December of 2013, serious, serious breaches in the FDA mad cow feed ban have
been documented. The 2004 enhanced BSE surveillance program was so flawed, that
one of the top TSE prion Scientist for the CDC, Dr. Paul Brown stated ; Brown,
who is preparing a scientific paper based on the latest two mad cow cases to
estimate the maximum number of infected cows that occurred in the United States,
said he has "absolutely no confidence in USDA tests before one year ago" because
of the agency's reluctance to retest the Texas cow that initially tested
positive. see ; http://www.upi.com/Health_News/2006/03/15/Analysis-What-that-mad-cow-means/UPI-12841142465253/
The BSE surveillance and testing have also been proven to be flawed, and
the GAO and OIG have both raised serious question as to just how flawed it has
been (see GAO and OIG reports). North America has more documented TSE prion
disease, in different documented species (excluding the Zoo BSE animals in the
EU), then any other place on the Globe. This does not include the very
likelihood that TSE prion disease in the domestic feline and canine have been
exposed to high doses of the TSE prion disease vid pet food. To date, it’s still
legal to include deer from cwd zone into pet food or deer food. Specified Risk
Material i.e. SRM bans still being breach, as recently as just last month.
nvCJD or what they now call vCJD, another case documented in Texas last
month, with very little information being released to the public on about this
case? with still the same line of thought from federal officials, ‘it can’t
happen here’, so another vCJD blamed on travel of a foreign animal disease from
another country, while ignoring all the BSE TSE Prion risk factors we have here
in the USA and Canada, and the time that this victim and others, do spend in the
USA, and exposed to these risk factors, apparently do not count in any way with
regard to risk factor. a flawed process of risk assessment.
sporadic CJD, along with new TSE prion disease in humans, of which the
young are dying, of which long duration of illness from onset of symptoms to
death have been documented, only to have a new name added to the pot of prion
disease i.e. sporadic GSS, sporadic FFI, and or VPSPR. I only ponder how a
familial type disease could be sporadic with no genetic link to any family
member? when the USA is the only documented Country in the world to have
documented two different cases of atypical H-type BSE, with one case being
called atypical H-G BSE with the G meaning Genetic, with new science now showing
that indeed atypical H-type BSE is very possible transmitted to cattle via oral
transmission (Prion2014). sporadic CJD and VPSPR have been rising in Canada,
USA, and the UK, with the same old excuse, better surveillance. You can only use
that excuse for so many years, for so many decades, until one must conclude that
CJD TSE prion cases are rising. a 48% incease in CJD in Canada is not just a
blip or a reason of better surveillance, it is a mathematical rise in numbers.
More and more we are seeing more humans exposed in various circumstance in the
Hospital, Medical, Surgical arenas to the TSE Prion disease, and at the same
time in North America, more and more humans are becoming exposed to the TSE
prion disease via consumption of the TSE prion via deer and elk, cattle, sheep
and goats, and for those that are exposed via or consumption, go on to further
expose many others via the iatrogenic modes of transmission of the TSE prion
disease i.e. friendly fire. I pondered this mode of transmission via the victims
of sporadic FFI, sporadic GSS, could this be a iatrogenic event from someone
sub-clinical with sFFI or sGSS ? what if?
Two decades have passed since Dr. Ironside first confirmed his first ten
nvCJD victims in 1995. Ten years later, 2005, we had Dr. Gambetti and his first
ten i.e. VPSPR in younger victims. now we know that indeed VPSPR is
transmissible. yet all these TSE prion disease and victims in the USA and Canada
are being pawned off as a spontaneous event, yet science has shown, the
spontaneous theory has never been proven in any natural case of TSE prion
disease, and scientist have warned, that they have now linked some sporadic CJD
cases to atypical BSE, to atypical Scrapie, and to CWD, yet we don’t here about
this in the public domain. We must make all human and animal TSE prion disease
reportable in every age group, in ever state and internationally, we must have a
serious re-evaluation and testing of the USA cattle herds, and we must ban
interstate movement of all cervids. Any voluntary effort to do any of this will
fail. Folks, we have let the industry run science far too long with regards to
the TSE prion disease. While the industry and their lobbyist continues to funnel
junk science to our decision policy makers, Rome burns. ...end
REFERENCES
[all scientific peer review studies and other scientific information I have
put into blogs, to shorten reference data. I DO NOT advertise or make money from
this, this information is for education use...lost my mom to the hvCJD, and just
made a promise, never forget, and never let them forget. ...TSS]
SNIP...SEE FULL TEXT ;
Sunday, June 29, 2014
*** Transmissible Spongiform Encephalopathy TSE Prion Disease North America
2014
Saturday, June 14, 2014
Rep. Rosa DeLauro (D-CT) Calls for Briefing on Beef Recalled for Mad Cow
Potential Rep. Rosa DeLauro (D-CT)
Thursday, June 12, 2014
Missouri Firm Recalls Ribeye and Carcass Products That May Contain
Specified Risk Materials 4,012 pounds of fresh beef products because the dorsal
root ganglia may not have been completely removed
Monday, June 02, 2014
Confirmed Human BSE aka mad cow Variant CJD vCJD or nvCJD Case in Texas
Monday, June 9, 2014
TEXAS MAD COW COVER UP (human BSE) AGAIN IN TEXAS, Mr. President Sir, we
need your help please
The most recent assessments (and reassessments) were published in June 2005
(Table I; 18), and included the categorisation of Canada, the USA, and Mexico as
GBR III. Although only Canada and the USA have reported cases, the historically
open system of trade in North America suggests that it is likely that BSE is
present also in Mexico.
IN SHORT, AND IN A NUT SHELL ;
(Adopted by the International Committee of the OIE on 23 May 2006)
11. Information published by the OIE is derived from appropriate
declarations made by the official Veterinary Services of Member Countries. The
OIE is not responsible for inaccurate publication of country disease status
based on inaccurate information or changes in epidemiological status or other
significant events that were not promptly reported to the Central Bureau,
Wednesday, February 12, 2014
USDA/APHIS NOTICE: Final Rule Regarding Imports and BSE Effective March 4,
2014
Sunday, July 06, 2014
Dietary Risk Factors for Sporadic Creutzfeldt-Jakob Disease: A Confirmatory
Case-Control Study
Conclusions—The a priori hypotheses were supported.
*Consumption of various meat products may be one method of transmission of
the infectious agent for sCJD.
TSS
