# Detection of Prion Infectivity in Fat Tissues of Scrapie-Infected Mice



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Detection of Prion Infectivity in Fat Tissues of Scrapie-Infected Mice 

Brent Race1#, Kimberly Meade-White1#, Michael B. A. Oldstone2, Richard Race1, Bruce Chesebro1*

1 Laboratory of Persistent Virus Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America, 2 Department of Immunology and Microbial Science, The Scripps Research Institute, LaJolla, California, United States of America

Abstract 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.

Author Summary 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. We show for the first time the presence of prion infectivity in white fat and brown fat tissues of mice with prion disease. Our results suggest that fat tissues of domestic or wild animals infected with prions may pose an unappreciated hazard for spread of infection to humans or domestic animals. The presence of prion infectivity in fat suggests that additional consideration may be required to eliminate from the food chain any fat from ruminants suspected of exposure to or infection with prions. Thus, this finding has implications for public health, food safety, and prion disease prevention strategies.

Citation: Race B, Meade-White K, Oldstone MBA, Race R, Chesebro B (2008) Detection of Prion Infectivity in Fat Tissues of Scrapie-Infected Mice. PLoS Pathog 4(12): e1000232. doi:10.1371/journal.ppat.1000232

Editor: Neil Mabbott, University of Edinburgh, United Kingdom

Received: August 12, 2008; Accepted: November 5, 2008; Published: December 5, 2008

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

Funding: This research was supported in part by the Intramural Research Program of the NIH, NIAID. MBAO was funded through NIA grant AG04032.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

# These authors contributed equally to this work.

Introduction

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Discussion The present results indicate that white fat and brown fat are possible tissue sources of prion infectivity which might play a role in transmission of prion disease. In vivo brown fat has a limited distribution, usually found in young animals in the intrascapular region and around various organs such as heart and kidney. In adult ruminants brown fat is minimal. Therefore, brown fat from infected animals is unlikely to be consumed by humans in large amounts. In contrast, humans often consume large amounts of ruminant white fat. In premium cuts of meat containing mostly skeletal muscle, white fat is often intertwined with muscle cells, and it is impossible to separate the two cell types. However, white fat, free of muscle, is found in subcutaneous, retroperitoneal, intraperitoneal, perirenal and other regions. Such fat is used in many processed meat products such as sausages and canned meats, and is also used in animal feeds. Our present data show clearly that fat in the absence of muscle has significant infectivity titers, which are similar to titers in muscle containing fat (Table 1). Since our skeletal muscle samples are unavoidably contaminated by white fat, it is possible that fat might be a contributor to the infectivity found in muscle. In support of this possibility we found PrPres detectable by IHC at high levels in white fat associated with skeletal muscle in some tg44 mice (Figure 4). In contrast, other groups did not mention seeing PrPres in muscle-associated fat tissue in animals where myocytes themselves were seen to be positive by IHC [13]-[20].

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It is unclear why there is accumulation of PrPres and infectivity in adipose tissues. One possibility might be the high level of innervation by the autonomic nervous system in both brown and white fat. In WT mice, nerves should express cell membrane anchored PrPC (PrPsen). Sympathetic nerves have been previously implicated in transfer of scrapie infectivity from spleen to brain in mice [29], and they might also play a role in infection of fat in WT mice. In tg44 mice the mechanism of fat infection is likely to be different as there is no anchored PrPsen on the nerves. We currently postulate a role for connective tissue structures in this process.

Infectivity in fat might also contribute to environmental contamination following the death of prion infected animals. Although infectivity titers are lower in fat and muscle than in CNS, the large mass of fat and muscle makes the total infectivity from these sources similar. Furthermore, fat and muscle are readily accessible to the environment after death, whereas the CNS is highly confined in skull and vertebral column. These factors might increase the importance of fat and muscle as sources of spread of prion disease among animals.

The low or negative plasma titers found in tg44 and WT mice indicate that residual plasma cannot account for the high infectivity levels seen in fat and other tissues (Table 1). However, low levels of plasma or blood-borne infectivity might still be a mechanism for spread of infectivity among tissues in tg44 mice and possibly also WT mice. Similarly transmission of low level blood prion infectivity has been documented by blood transfusion in BSE-infected sheep [30], and also accounts for some rare cases of human variant CJD [31],[32].

In this study extraneural infection was much higher in tg44 mice expressing anchorless PrP than in WT mice. The explanation of this finding is unclear. Possibly soluble anchorless PrP facilitates spread of infection from CNS to extraneural sites by blood, lymph or nerve-mediated transport. Alternatively, the long asymptomatic survival time of tg44 mice might also contribute to high level extraneural infection. This could also be a factor in many animal prion diseases where the time course is long, i.e. 2-5 years or more, and might allow higher extraneural infectivity in fat tissues [7], [33]-[35].

The present data using a mouse model shows the proof of principle that brown and white fat tissues can be important sites of prion agent deposition. It will be important to extend these studies in the future to prion infected large animals such as cattle, sheep and cervids where there may be greater potential for contamination of human or domestic animal food chains. We are currently doing this experiment with fat from CWD deer; however, it will require an additional year to gather this data, and this result is therefore beyond the scope of the present paper. Such studies may be difficult because of the lower titers seen in these large animals compared to rodent scrapie models. For example, we often detect titers of 9-10 logID50/gram of mouse brain, whereas in brain from BSE cattle [8], and scrapie sheep [4] titers reported are 7-8 logID50/gram. We are finding similar low titers in CWD cervid brain in our deer PrP transgenic mice (unpublished data). These results could indicate either that the amount of prion agent present in ruminant brain is lower than in mice and hamsters or that the cattle, sheep and deer PrP transgenic mice used for infectivity assays are less sensitive than the WT mice or hamster PrP transgenic mice used for rodent scrapie. In either case this might affect ability to detect infectivity in fat of these important large animal models.

Materials and Methods

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http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000232


Evaluation of the Human Transmission Risk of an Atypical Bovine Spongiform Encephalopathy Prion Strain

Qingzhong Kong,1* Mengjie Zheng,1 Cristina Casalone,2 Liuting Qing,1 Shenghai Huang,1? Bikram Chakraborty,1 Ping Wang,1 Fusong Chen,1 Ignazio Cali,1 Cristiano Corona,2 Francesca Martucci,2 Barbara Iulini,2 Pierluigi Acutis,2 Lan Wang,1 Jingjing Liang,1 Meiling Wang,1 Xinyi Li,1 Salvatore Monaco,3 Gianluigi Zanusso,3 Wen-Quan Zou,1 Maria Caramelli,2 and Pierluigi Gambetti1* 
Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106,1 CEA, Istituto Zooprofilattico Sperimentale, 10154 Torino, Italy,2 Department of Neurological and Visual Sciences, University of Verona, 37134 Verona, Italy3
*Corresponding author. Mailing address: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106. Phone for Pierluigi Gambetti: (216) 368-0586. Fax: (216) 368-2546. E-mail: [email protected] . Phone for Qingzhong Kong: (216) 368-1756. Fax: (216) 368-2546. E-mail: [email protected] 
?Present address: Department of Patient Education and Health Information, Cleveland Clinic Foundation, Cleveland, OH 44195.
Received November 30, 2007; Accepted January 16, 2008.

Bovine spongiform encephalopathy (BSE), the prion disease in cattle, was widely believed to be caused by only one strain, BSE-C. BSE-C causes the fatal prion disease named new variant Creutzfeldt-Jacob disease in humans. Two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H, have been discovered in several countries since 2004; their transmissibility and phenotypes in humans are unknown. We investigated the infectivity and human phenotype of BASE strains by inoculating transgenic (Tg) mice expressing the human prion protein with brain homogenates from two BASE strain-infected cattle. Sixty percent of the inoculated Tg mice became infected after 20 to 22 months of incubation, a transmission rate higher than those reported for BSE-C. A quarter of BASE strain-infected Tg mice, but none of the Tg mice infected with prions causing a sporadic human prion disease, showed the presence of pathogenic prion protein isoforms in the spleen, indicating that the BASE prion is intrinsically lymphotropic. The pathological prion protein isoforms in BASE strain-infected humanized Tg mouse brains are different from those from the original cattle BASE or sporadic human prion disease. Minimal brain spongiosis and long incubation times are observed for the BASE strain-infected Tg mice. These results suggest that in humans, the BASE strain is a more virulent BSE strain and likely lymphotropic.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2268471


Thursday, December 04, 2008 2:37 PM

"we have found that H-BSE can infect humans."

personal communication with Professor Kong. ...TSS



November 25, 2008

Update On Feed Enforcement Activities To Limit The Spread Of BSE

http://madcowfeed.blogspot.com/2008/11/november-2008-update-on-feed.html


"the biochemical signature of PrPres in the BASE-inoculated animal was found to have a higher proteinase K sensitivity of the octa-repeat region. We found the same biochemical signature in three of four human patients with sporadic CJD and an MM type 2 PrP genotype who lived in the same country as the infected bovine."

just another one of those sporadic CJD coincidences i suppose $$$

NOT to forget ;

Thursday, June 05, 2008

Review on the epidemiology and dynamics of BSE epidemics

Vet. Res. (2008) 39:15 www.vetres.org DOI: 10.1051/vetres:2007053 c INRA, EDP Sciences, 2008 Review article

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And last but not least, similarities of PrPres between Htype BSE and human prion diseases like CJD or GSS have been put forward [10], as well as between L-type BSE and CJD [17]. These findings raise questions about the origin and inter species transmission of these prion diseases that were discovered through the BSE active surveillance.

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Cases of atypical BSE have only been found in countries having implemented large active surveillance programs. As of 1st September 2007, 36 cases (16 H, 20 L) have been described all over the world in cattle: Belgium (1 L) [23], Canada (1 H)15, Denmark (1 L)16, France (8 H, 6 L)17, Germany (1 H, 1 L) [13], Italy (3 L)18, Japan (1 L) [71], Netherlands (1 H, 2 L)19, Poland (1 H, 6 L)20, Sweden (1 H)21, United Kingdom (1 H)22, and USA (2 H)23. Another H-type case has been found in a 19 year old miniature zebu in a zoological park in Switzerland [56]. It is noteworthy that atypical cases have been found in countries that did not experience classical BSE so far, like Sweden, or in which only few cases of classical BSE have been found, like Canada or the USA.

And last but not least, similarities of PrPres between Htype BSE and human prion diseases like CJD or GSS have been put forward [10], as well as between L-type BSE and CJD [17]. These findings raise questions about the origin and inter species transmission of these prion diseases that were discovered through the BSE active surveillance.

full text 18 pages ;

http://www.vetres.org/index.php?opt...9&url=/articles/vetres/pdf/2008/04/v07232.pdf

please see full text ;

http://bse-atypical.blogspot.com/2008/06/review-on-epidemiology-and-dynamics-of.html

***Atypical forms of BSE have emerged which, although rare, appear to be more virulent than the classical BSE that causes vCJD.***

Progress Report from the National Prion Disease Pathology Surveillance Center

An Update from Stephen M. Sergay, MB, BCh & Pierluigi Gambetti, MD

April 3, 2008

http://www.aan.com/news/?event=read&article_id=4397&page=72.45.45

Sunday, March 16, 2008

MAD COW DISEASE terminology UK c-BSE (typical), atypical BSE H or L, and or Italian L-BASE

http://bse-atypical.blogspot.com/2008/03/mad-cow-disease-terminology-uk-c-bse.html

HUMAN and ANIMAL TSE Classifications i.e. mad cow disease and the UKBSEnvCJD only theory JUNE 2008

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Tissue infectivity and strain typing of the many variants Manuscript of the human and animal TSEs are paramount in all variants of all TSE. There must be a proper classification that will differentiate between all these human TSE in order to do this. With the CDI and other more sensitive testing coming about, I only hope that my proposal will some day be taken seriously. ...

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http://cjdmadcowbaseoct2007.blogspot.com/2008/06/human-and-animal-tse-classifications-ie.html


see full text ;


Friday, December 05, 2008

Detection of Prion Infectivity in Fat Tissues of Scrapie-Infected Mice 

http://scrapie-usa.blogspot.com/2008/12/detection-of-prion-infectivity-in-fat.html


Monday, December 08, 2008

vCJD & dental treatment 

http://creutzfeldt-jakob-disease.blogspot.com/2008/12/vcjd-dental-treatment.html



Monday, December 8, 2008

Genetic risk factors for variant Creutzfeldt-Jakob disease: a genome-wide association study


http://sporadicffi.blogspot.com/2008/12/genetic-risk-factors-for-variant.html




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