Monday, July 05, 2010

Immunohistochemical Detection of Disease-Associated Prion Protein in the Intestine of Cattle Naturally Affected with Bovine Spongiform Encephalopathy

Immunohistochemical Detection of Disease-Associated Prion Protein in the Intestine of Cattle Naturally Affected with Bovine Spongiform Encephalopathy by Using an Alkaline-Based Chemical Antigen Retrieval Method

Hiroyuki OKADA1), Yoshihumi IWAMARU1), Morikazu IMAMURA1), Kentaro MASUJIN1), Takashi YOKOYAMA1) and Shirou MOHRI1)

1) Prion Disease Research Center, National Institute of Animal Health

(Received 18-May-2010) (Accepted 17-Jun-2010)

ABSTRACT. An alkaline-based chemical antigen retrieval pretreatment step was used to enhance immunolabeling of disease-associated prion protein (PrPSc) in formalin-fixed and paraffin-embedded tissue sections from cattle naturally affected with bovine spongiform encephalopathy (BSE). The modified chemical method used in this study amplified the PrPSc signal by unmasking PrPSc compared with the normal cellular prion protein. In addition, this method reduced nonspecific background immunolabeling that resulted from the destruction of the residual normal cellular form of prion protein, and reduced the treatment time compared with the usual autoclave pretreatment step. Immunolabeled PrPSc was thereby clearly detected in the myenteric plexus of the ileum in naturally occurring BSE cattle.

KEY WORDS: antigen retrieval, BSE, cattle, immunohistochemistry, prion

This is, to the best of our knowledge, the first description of the localization of PrPSc within the colon of naturally affected BSE cattle. In the present study, PrPSc was observed in the myenteric plexus of the ileum and colon, but not in the duodenum and jejunum, using immunohistochemistry, western blot analysis, and mouse bioassay (Table 3). PrPSc has been detected in the myenteric plexus of the ileum from naturally affected BSE cattle [10, 12], chronic wasting disease in deer [22], and sheep scrapie [1], Based on the current BSE research, PrPSc is not found in gastrointestinal tissues other than the distal ileum [27]. BSE infectivity of the peripheral nervous system (PNS) was found in one clinical case [4]. PrPSc accumulation in the PNS of BSE-affected cattle during the clinical stages of infection may be the rule in BSE rather than the exception; it also may be the main mechanism rather than merely preceding the accumulation in the central nervous system (CNS) [14]. Results from western blotting and mouse bioassay indicated relatively low amounts of PrPSc deposits in the ileum and colon. 12 These data suggest that detectable PrPSc in the ileum and colon is probably caused by centrifugal spread from the CNS to the ENS within the peripheral nerves during the clinical stage of the disease. Therefore, to determine whether PrPSc was present in the ENS, the corresponding tissues should be subjected to western blot analysis by using the highly sensitive protocols described in this study or analyzed with a more sensitive mouse bioassay, or both.

In summary, chemical pretreatment with NaOH may provide a useful procedure for the detection of immunolabeled PrPSc and the suppression of the remaining PK-resistant PrPC without any nonspecific background immunostaining in FFPE tissue sections from BSE-affected cattle compared to the usual autoclave pretreatment step. In addition, pretreatment using NaOH at a higher temperature in the alkaline-based chemical antigen retrieval method clearly enhanced the strength of the immunolabeled PrPSc signal.

Thursday, June 24, 2010

Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues

Volume 16, Number 7–July 2010

To date the OIE/WAHO assumes that the human and animal health standards set out in the BSE chapter for classical BSE (C-Type) applies to all forms of BSE which include the H-type and L-type atypical forms. This assumption is scientifically not completely justified and accumulating evidence suggests that this may in fact not be the case. Molecular characterization and the spatial distribution pattern of histopathologic lesions and immunohistochemistry (IHC) signals are used to identify and characterize atypical BSE. Both the L-type and H-type atypical cases display significant differences in the conformation and spatial accumulation of the disease associated prion protein (PrPSc) in brains of afflicted cattle. Transmission studies in bovine transgenic and wild type mouse models support that the atypical BSE types might be unique strains because they have different incubation times and lesion profiles when compared to C-type BSE. When L-type BSE was inoculated into ovine transgenic mice and Syrian hamster the resulting molecular fingerprint had changed, either in the first or a subsequent passage, from L-type into C-type BSE. In addition, non-human primates are specifically susceptible for atypical BSE as demonstrated by an approximately 50% shortened incubation time for L-type BSE as compared to C-type. Considering the current scientific information available, it cannot be assumed that these different BSE types pose the same human health risks as C-type BSE or that these risks are mitigated by the same protective measures.

14th International Congress on Infectious Diseases H-type and L-type Atypical BSE January 2010 (special pre-congress edition)

18.173 page 189

Experimental Challenge of Cattle with H-type and L-type Atypical BSE

A. Buschmann1, U. Ziegler1, M. Keller1, R. Rogers2, B. Hills3, M.H. Groschup1. 1Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany, 2Health Canada, Bureau of Microbial Hazards, Health Products & Food Branch, Ottawa, Canada, 3Health Canada, Transmissible Spongiform Encephalopathy Secretariat, Ottawa, Canada

Background: After the detection of two novel BSE forms designated H-type and L-type atypical BSE the question of the pathogenesis and the agent distribution of these two types in cattle was fully open. From initial studies of the brain pathology, it was already known that the anatomical distribution of L-type BSE differs from that of the classical type where the obex region in the brainstem always displays the highest PrPSc concentrations. In contrast in L-type BSE cases, the thalamus and frontal cortex regions showed the highest levels of the pathological prion protein, while the obex region was only weakly involved.

Methods:We performed intracranial inoculations of cattle (five and six per group) using 10%brainstemhomogenates of the two German H- and L-type atypical BSE isolates. The animals were inoculated under narcosis and then kept in a free-ranging stable under appropriate biosafety conditions.At least one animal per group was killed and sectioned in the preclinical stage and the remaining animals were kept until they developed clinical symptoms. The animals were examined for behavioural changes every four weeks throughout the experiment following a protocol that had been established during earlier BSE pathogenesis studies with classical BSE.

Results and Discussion: All animals of both groups developed clinical symptoms and had to be euthanized within 16 months. The clinical picture differed from that of classical BSE, as the earliest signs of illness were loss of body weight and depression. However, the animals later developed hind limb ataxia and hyperesthesia predominantly and the head. Analysis of brain samples from these animals confirmed the BSE infection and the atypical Western blot profile was maintained in all animals. Samples from these animals are now being examined in order to be able to describe the pathogenesis and agent distribution for these novel BSE types. Conclusions: A pilot study using a commercially avaialble BSE rapid test ELISA revealed an essential restriction of PrPSc to the central nervous system for both atypical BSE forms. A much more detailed analysis for PrPSc and infectivity is still ongoing.

14th ICID International Scientific Exchange Brochure -

Final Abstract Number: ISE.114

Session: International Scientific Exchange

Transmissible Spongiform encephalopathy (TSE) animal and human TSE in North America

update October 2009

T. Singeltary

Bacliff, TX, USA


An update on atypical BSE and other TSE in North America. Please remember, the typical U.K. c-BSE, the atypical l-BSE (BASE), and h-BSE have all been documented in North America, along with the typical scrapie's, and atypical Nor-98 Scrapie, and to date, 2 different strains of CWD, and also TME. All these TSE in different species have been rendered and fed to food producing animals for humans and animals in North America (TSE in cats and dogs ?), and that the trading of these TSEs via animals and products via the USA and Canada has been immense over the years, decades.


12 years independent research of available data


I propose that the current diagnostic criteria for human TSEs only enhances and helps the spreading of human TSE from the continued belief of the UKBSEnvCJD only theory in 2009. With all the science to date refuting it, to continue to validate this old myth, will only spread this TSE agent through a multitude of potential routes and sources i.e. consumption, medical i.e., surgical, blood, dental, endoscopy, optical, nutritional supplements, cosmetics etc.


I would like to submit a review of past CJD surveillance in the USA, and the urgent need to make all human TSE in the USA a reportable disease, in every state, of every age group, and to make this mandatory immediately without further delay. The ramifications of not doing so will only allow this agent to spread further in the medical, dental, surgical arena's. Restricting the reporting of CJD and or any human TSE is NOT scientific. Iatrogenic CJD knows NO age group, TSE knows no boundaries. I propose as with Aguzzi, Asante, Collinge, Caughey, Deslys, Dormont, Gibbs, Gajdusek, Ironside, Manuelidis, Marsh, et al and many more, that the world of TSE Transmissible Spongiform Encephalopathy is far from an exact science, but there is enough proven science to date that this myth should be put to rest once and for all, and that we move forward with a new classification for human and animal TSE that would properly identify the infected species, the source species, and then the route.

International Society for Infectious Diseases Web:

I ask Professor Kong ;

Thursday, December 04, 2008 3:37 PM Subject: RE: re--Chronic Wating Disease (CWD) and Bovine Spongiform Encephalopathies (BSE): Public Health Risk Assessment

''IS the h-BSE more virulent than typical BSE as well, or the same as cBSE, or less virulent than cBSE? just curious.....''

Professor Kong reply ;


''As to the H-BSE, we do not have sufficient data to say one way or another, but we have found that H-BSE can infect humans. I hope we could publish these data once the study is complete.

Thanks for your interest.''

Best regards,

Qingzhong Kong, PhD Associate Professor Department of Pathology Case Western Reserve University Cleveland, OH 44106 USA



Molecular Features of the Protease-resistant Prion Protein (PrPres) in H-type BSE

Biacabe, A-G1; Jacobs, JG2; Gavier-Widén, D3; Vulin, J1; Langeveld, JPM2; Baron, TGM1 1AFSSA, France; 2CIDC-Lelystad, Netherlands; 3SVA, Sweden

Western blot analyses of PrPres accumulating in the brain of BSE-infected cattle have demonstrated 3 different molecular phenotypes regarding to the apparent molecular masses and glycoform ratios of PrPres bands. We initially described isolates (H-type BSE) essentially characterized by higher PrPres molecular mass and decreased levels of the diglycosylated PrPres band, in contrast to the classical type of BSE. This type is also distinct from another BSE phenotype named L-type BSE, or also BASE (for Bovine Amyloid Spongiform Encephalopathy), mainly characterized by a low representation of the diglycosylated PrPres band as well as a lower PrPres molecular mass. Retrospective molecular studies in France of all available BSE cases older than 8 years old and of part of the other cases identified since the beginning of the exhaustive surveillance of the disease in 20001 allowed to identify 7 H-type BSE cases, among 594 BSE cases that could be classified as classical, L- or H-type BSE. By Western blot analysis of H-type PrPres, we described a remarkable specific feature with antibodies raised against the C-terminal region of PrP that demonstrated the existence of a more C-terminal cleaved form of PrPres (named PrPres#2 ), in addition to the usual PrPres form (PrPres #1). In the unglycosylated form, PrPres #2 migrates at about 14 kDa, compared to 20 kDa for PrPres #1. The proportion of the PrPres#2 in cattle seems to by higher compared to the PrPres#1. Furthermore another PK–resistant fragment at about 7 kDa was detected by some more N-terminal antibodies and presumed to be the result of cleavages of both N- and C-terminal parts of PrP. These singular features were maintained after transmission of the disease to C57Bl/6 mice. The identification of these two additional PrPres fragments (PrPres #2 and 7kDa band) reminds features reported respectively in sporadic Creutzfeldt-Jakob disease and in Gerstmann-Sträussler-Scheinker (GSS) syndrome in humans.

Wednesday, March 31, 2010

Atypical BSE in Cattle

Saturday, June 12, 2010

PUBLICATION REQUEST AND FOIA REQUEST Project Number: 3625-32000-086-05 Study of Atypical Bse

Archive Number 20100405.1091 Published Date 05-APR-2010

Subject PRO/AH/EDR> Prion disease update 1010 (04)


[Terry S. Singeltary Sr. has added the following comment:

"According to the World Health Organisation, the future public health threat of vCJD in the UK and Europe and potentially the rest of the world is of concern and currently unquantifiable. However, the possibility of a significant and geographically diverse vCJD epidemic occurring over the next few decades cannot be dismissed


The key word here is diverse. What does diverse mean? If USA scrapie transmitted to USA bovine does not produce pathology as the UK c-BSE, then why would CJD from there look like UK vCJD?",F2400_P1001_PUB_MAIL_ID:1000,82101

> Up until about 6 years ago, the pt worked at Tyson foods where she

> worked on the assembly line, slaughtering cattle and preparing them for

> packaging. She was exposed to brain and spinal cord matter when she

> would euthanize the cattle.


Monday, April 5, 2010


Tuesday, June 1, 2010

USA cases of dpCJD rising with 24 cases so far in 2010

Wednesday, June 16, 2010

Defining sporadic Creutzfeldt-Jakob disease strains and their transmission properties


Tuesday, March 2, 2010

Animal Proteins Prohibited in Ruminant Feed/Adulterated/Misbranded Rangen Inc 2/11/10 USA

Monday, March 1, 2010


Terry S. Singeltary Sr. (Submitted question): Monday, April 5, 2010

Update on Feed Enforcement Activities to Limit the Spread of BSE April 5, 2010


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