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Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease

Posted Sep 07 2008 2:06am
Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease

Beiru Zhang*†, Yumi Une‡, Xiaoying Fu*, Jingmin Yan*, FengXia Ge*, Junjie Yao*§, Jinko Sawashita*, Masayuki Mori*, Hiroshi Tomozawa¶, Fuyuki Kametani, and Keiichi Higuchi*‡** *Department of Aging Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, and ¶Division of Laboratory Animal Research, Research Center for Human and Environmental Science, Shinshu University, 3-1-1, Asahi, Matsumoto 390-8621, Japan; †Department of Nephrology, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, China; ‡Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 229-8501, Japan; §The Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo 183-8508, Japan; and Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, Tokyo 156-8585, Japan Edited by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved April 1, 2008 (received for review January 16, 2008)

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


Discussion It is currently accepted that systemic AA amyloidosis is an increasingly important cause of morbidity and mortality in captive cheetah populations (14). For conservation of this species, therefore, it is critical to elucidate the etiology of AA amyloidosis. As with sheep scrapie and cervid CWD, the routes of transmission are among the most debated and intriguing issues. InfectiousCWDprions in saliva have been identified to be involved in transmission in high-density captive situations (19, 20). Recently, available evidence indicates that an environmental reservoir of infectivity contributes to the continuation of these diseases in affected populations. These infectious agents can be transmitted by flesh flies (21) or hay mites (22) and can directly enter the environment from decomposing carcasses of infected animals (23). Environmental contamination by excreta from infected cervids has also seemed the most plausible explanation for the dissemination of CWD (24). Scrapie-infected hamsters and Creutzfeldt–Jakob disease (CJD) patients were reported to excrete urinary protease-resistant PrP isoform (25), indicating that urinary excretion from infected animals may provide a vector for horizontal transmission. However, there are studies that are not consistent with these findings (26, 27). Perhaps unrecognized nephritic conditions may underlie these discrepant observations, because it has been reported that urinary prion excretion is found only in scrapie-infected mice with lymphocytic nephritis (28). In this study, we observed several bands with high molecular weights that reacted with anti-cheetah AA antiserum in the whole urine sample, but not in the urine pellet in whichAAamyloid fibrils should be recovered. We thought that the possibility for a transmission pathway through urine might be low, but it could not be ruled out. In addition to urine, the alimentary shedding route has been considered as a possible transmission pathway (29). Abnormal prion protein is present in gut-associated lymphoid tissues of mule deer infected with CWD, consistent with an alimentary shedding route (30). In this study, we showed that the fecal fraction from a cheetah with amyloidosis had AA amyloid fibrils and possessed high transmissibility. In mouse AApoAII amyloidosis, regarded recently as another transmissible amyloidosis (5–7), we also demonstrated that the feces could serve as an agent to induce amyloidosis in recipient mice (31). These results shed new light on the etiology involved in the high incidence of AA amyloidosis in cheetahs. In this study, we unexpectedly found that the amyloid fibril fraction from feces had smaller amyloid fibrils and higher sensitivity to denaturation treatment than the liver amyloid fibril fraction. In mammalian prion, it has been demonstrated that there is a very strong correlation between seeding capability and amyloid fibril conformation (32, 33). Similarly, in yeast prion, it also has been indicated that [PSI] with stronger infectivity typically have less stable fibrils in vivo than strains with weaker infectivity (34), and the prion strain with relatively smaller prion particles is always associated with greater frangibility and increased sensitivity to denaturants (35). The enhanced frangibility is presumably involved in the increase in seeding efficiency and prion infectivity, while the high sensitivity probably results from structural differences in inter-molecular contacts and a shorter, less stable amyloid core. The divergent ultrastructure between the fecal and the liver fibrils identified by transmission electron microscopy may be responsible for the different characteristics of transmissibility and sensitivity to denaturation treatment, analogous to prion protein. It has been reported that AA amyloidosis can be experimentally induced by i.v. or i.p. administration of AA amyloid fibrillar extracts in recipient mice (10). A few recent studies have shown that AA-containing extracts also had amyloid-inducing activity when administered orally to mice (36, 37). In AApoAII amyloidosis, we eported that an oral administration of AApoAII amyloid fibrils induced amyloidosis in recipient mice (38). Thus, it is plausible that oral ingestion of AA-containing fecal matter caused amyloid deposition in the cheetah population. At this juncture, the manner in which fecal matter is initially absorbed by the cheetahs is not clear. This may occur during mutual grooming (licking of the fur contaminated by fecal matter). Recently it was shown that a prion agent could bind to whole soil and common soil minerals and retain infectivity for a prolonged period (23, 39). Thus, soil may act as a reservoir capable of contaminating both food and fur. It is also unknown how AA fibril proteins enter the feces. Because AA amyloidosis was also in the small intestines of AA amyloidosis cheetahs, it is possible that AA proteins enter the feces through exfoliated mucosa. In conclusion, we found that cheetahs with amyloidosis pass fecal matter that had strong seeding efficiency and should be regarded as a transmission medium. To control the incidence of AA amyloidosis and reduce the likelihood of the animal’s extinction, prevention of the transmission with excretion from cheetahs with amyloidosis should be considered along with reduction of precursor SAA levels.

Materials and Methods


Wednesday, August 13, 2008 Excretion of BSE and scrapie prions in stools from murine models


Subject: Infectious Prions in the Saliva and Blood of Deer with Chronic Wasting Disease

Date: October 5, 2006 at 1:45 pm PST

Infectious Prions in the Saliva

and Blood of Deer with Chronic

Wasting Disease

Candace K. Mathiason,1 Jenny G. Powers,3 Sallie J. Dahmes,4 David A. Osborn,5 Karl V. Miller,5

Robert J. Warren,5 Gary L. Mason,1 Sheila A. Hays,1 Jeanette Hayes-Klug,1 Davis M. Seelig,1

Margaret A. Wild,3 Lisa L. Wolfe,6 Terry R. Spraker,1,2 Michael W. Miller,6 Christina J. Sigurdson,1

Glenn C. Telling,7 Edward A. Hoover1*

A critical concern in the transmission of prion diseases, including chronic wasting disease (CWD) of cervids, is the potential presence of prions in body fluids. To address this issue directly, we exposed cohorts of CWD-nai¨ve deer to saliva, blood, or urine and feces from CWD-positive deer.

We found infectious prions capable of transmitting CWD in saliva (by the oral route) and in blood (by transfusion). The results help to explain the facile transmission of CWD among cervids and prompt caution concerning contact with body fluids in prion infections.


Deer cohorts 1 (blood), 2 (saliva), and 3 (urine and feces) were electively euthanized at 18 months pi to permit whole-body examination for PrPCWD. The greatest scrutiny was directed toward those tissues previously established to have highest frequency of PrPCWD deposition in infected deer and generally regarded as the most sensitive indicators of infection- medulla oblongata and other brainstem regions, tonsil, and retropharyngeal lymph node. We found unequivocal evidence of PrPCWD in brain and lymphoid tissue of all six tonsil biopsy- positive deer in cohorts 1 (blood) and 2 (saliva), whereas all deer in cohorts 3 and 5 were negative for PrPCWD in all tissues (Table 2 and

Figs. 1 and 2).

The transmission of CWD by a single blood transfusion from two symptomatic and one asymptomatic CWDþ donor is important in at least three contexts: (i) It reinforces that no tissue from CWD-infected cervids can be considered free of prion infectivity; (ii) it poses the possibility of hematogenous spread of CWD, such as through insects; and (iii) it provides a basis for seeking in vitro assays sufficiently sensitive to demonstrate PrPCWD or alternate prion protein conformers in blood-one of the grails of prion biology and epidemiology.

The identification of blood-borne prion transmission has been sought before with mixed results (9-11). Bovine spongiform encephalopathy and scrapie have been transmitted to naBve sheep through the transfer of 500 ml of blood or buffy coat white blood cells from infected sheep (12, 13). In addition, limited but compelling evidence argues for the transmission of variant Creutzfeldt-Jakob disease (vCJD) through blood from asymptomatic donors (14-16). Even in sporadic CJD, PrPres has been found in peripheral organs of some patients (17). The present work helps establish that prion diseases can be transmitted through blood.

The presence of infectious CWD prions in saliva may explain the facile transmission of CWD. Cervid-to-cervid interactions (SOM text), especially in high density and captive situations, would be expected to facilitate salivary crosscontact (11, 18, 19). Salivary dissemination of prions may not be limited to CWD. Proteaseresistant prion protein has been demonstrated in the oral mucosa, taste buds, lingual epithelium, vomeronasal organ, and olfactory mucosa of hamsters infected with transmissible mink encephalopathy (19) and ferrets infected with CWD (20). Although no instance of CWD transmission to humans has been detected, the present results emphasize the prudence of using impervious gloves during contact with saliva or blood of cervids that may be CWD-infected.

Environmental contamination by excreta from infected cervids has traditionally seemed the most plausible explanation for the dissemination of CWD (21). However, we could not detect PrPCWD in cohort 3 deer inoculated repeatedly with urine and feces from CWDþ deer and examined up to 18 months pi (Table 2). There are several reasons to view this negative finding cautiously, including small sample size, elective preclinical termination, and potential variation in individual susceptibility that may be associated with the 96 G/S polymorphism in the PRNP gene (7, 22). Although no genotype of white-tailed deer is resistant to CWD infection, PRNP genotypes S/S or G/S at codon 96 appear to have reduced susceptibility manifest by longer survival (7). Both deer in cohort 3 (urine and feces) were subsequently shown to be of the PRNP 96 G/S genotype. Thus, it is possible, although we think unlikely, that these deer had a prolonged incubation period (918 months pi) before the amplification of PrPCWD became detectable in tissues. Recent studies have shown that PrPres is poorly preserved after incubation with intestinal or fecal content (23, 24). Further research using cervid and surrogate cervid PrP transgenic mice (25) are indicated to continue to address the presence of infectious CWD prions in excreta of CWDþ deer and to provide a more substantial basis for reconsideration of the assumption that excreta are the chief vehicle for CWD dissemination and transmission.

The results reported here provide a plausible basis for the efficient transmission of CWD in nature. We demonstrate that blood and saliva in particular are able to transmit CWD to naBve deer and produce incubation periods consistent with those observed in naturally acquired infections (3, 26). The time from exposure to first detection of PrPCWD by tonsil biopsy was variable-as short as 3 months but as long as 18 months (likely underestimates due to sampling frequency). The results also reinforce a cautious view of the exposure risk presented by body fluids, excreta, and all tissues from CWDþ cervids. ...


CWD AND ENVIRONMENTAL FACTORS i.e. saliva, fecal shedding and fecal-oral transmission is likely

Chronic Wasting Disease CWD

Thursday, August 28, 2008

CWD TISSUE INFECTIVITY brain, lymph node, blood, urine, feces, antler velvet and muscle

Sunday, August 24, 2008 HAVE ANOTHER GLASS OF CWD PRIONS COURTESY Dane County Wisconsin Mike DiMaggio, solid waste manager

Thursday, August 28, 2008 cwd, feeding, and baiting piles

Tuesday, September 02, 2008 Detection of infectious prions in urine (Soto et al Available online 13 August 2008.)

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