Letter
Prion in Saliva of Bovine Spongiform Encephalopathy–Infected Cattle
To the Editor: A definitive diagnosis of bovine spongiform encephalopathy
(BSE) in cattle usually relies on Western blot and immunohistochemical testing
of samples from the obex region of the brainstem. These conventional diagnostic
tests can detect the presence of the abnormal (disease-associated) form of the
prion protein (PrPSc) in brain samples several months before the onset of
clinical signs; however, there is no appropriate, universal tool for early
preclinical and antemortem diagnosis of BSE. Furthermore, confirmation of the
disease is currently only possible by postmortem examination of brain tissues.
In this study, we used the serial protein misfolding cyclic amplification
(sPMCA) technique to determine the presence of PrPSc in saliva samples collected
from BSE-infected cows before and after the onset of disease (1). In a previous
study (2), we analyzed the tissue distribution of PrPSc in cattle up to 66
months after they were orally inoculated with a relatively low dose (5 g) of
homogenized brainstem from animals with naturally occurring BSE in England. In
2011, after publication of that study and 83.3 months after the cows were
inoculated, clinical signs of BSE developed in 1 cow (no. 5444); necropsy was
performed 84.7 months after inoculation. In addition, we used saliva samples
from 2 BSE-affected cows (nos. 5413 and 5437) (2) to determine the presence of
PrPSc. We collected saliva samples from animals at 4 monthly intervals,
beginning in 2009, 56 months after inoculation. Samples were stored at −80°C
until analysis. Using the sodium phosphotungstic acid precipitation method, we
concentrated (100-fold) individual 1-mL saliva samples from each time point. We
then diluted the concentrated samples 1:10 with the normal isoform of prion
protein substrate containing 0.5% potassium dextran sulfate. Using the sPMCA
technique as described (1), we amplified the samples in 3–8 tubes, and we used
Western blot to analyze the proteinase K–treated sPMCA products (2). Using
Western blot and immunohistochemical tests, we detected the accumulation of
PrPSc in brains collected at necropsy from the 3 cows examined. In addition,
using the sPMCA technique, we detected PrPSc signal in 1) saliva samples that
were concentrated from samples collected from the same 3 cows at necropsy and in
2) concentrated saliva samples that were collected from 2 of the cows (nos. 5413
and 5444) at the early clinical stages of disease.
Figure
Figure. . Western blot detection, using the serial protein misfolding
cyclic amplification technique, of the abnormal (disease-associated) form of the
prion protein (PrPSc) in concentrated saliva samples from 3 cows experimentally
infected...
After saliva samples underwent 3 rounds of amplification, we detected PrPSc
in a saliva sample that was collected from cow number 5437 two months before the
clinical onset of clinical symptoms (Figure). For 2 of the cows (nos. 5413 and
5437), the positive ratio of salivary PrPSc at round 4 of amplification
increased as the disease progressed (Figure). Because PrPSc signal could be
detected in BSE-infected brain homogenates diluted up to 10−10 after 2 rounds of
amplification (1), we estimated PrPSc levels in the nonconcentrated original
saliva samples to be lower than those in BSE-infected brain homogenate diluted
to 10−12. No PrPSc signal was detected in samples collected from the 3 cows 3–5
months before the onset of clinical symptoms or from age-matched noninfected
controls, even after 4 rounds of amplification. We demonstrated the presence of
PrPSc in saliva of BSE-affected cows during the clinical stage of the disease,
and in 1 case, at the preclinical or asymptomatic stage. Our findings suggest
that PrPSc is likely to be detected in the saliva of BSE-affected cattle during
the clinical stage of disease, after accumulation of PrPSc in the brain. PrPSc
was found in the salivary glands of BSE-affected cattle at the terminal stage of
infection (1). Therefore, once the infectious agent reaches the central nervous
system, it may spread centrifugally from the brain to the salivary glands
through the autonomic nervous system. Infectivity of saliva and the presence of
PrPSc in saliva have been reported in other ruminants affected with
transmissible spongiform encephalopathy. Infectivity of saliva was demonstrated
in deer with chronic wasting disease (3) and in scrapie-affected sheep (4); the
immunolabeled PrPSc accumulated in the salivary glands of scrapie-affected sheep
(5). A low level of PrPSc was detected in concentrated buccal swab samples of
preclinical scrapie-infected sheep by using sPMCA (6,7). These results suggest
that small amounts of PrPSc may accumulate in the salivary glands and are then
secreted into saliva. The presence of infectious prions in saliva may explain
the facile horizontal transmission of scrapie in sheep (4–6) and chronic wasting
disease in deer (4,8). There has been no epidemiologic evidence, however, that
saliva, milk, blood, and cerebrospinal fluid from BSE-infected cattle are
infectious (9). Nonetheless, the potential risk for BSE transmission by body
fluids or excretions from BSE-infected cattle is cannot be ruled out by the
current data.
Hiroyuki Okada, Yuichi Murayama , Noriko Shimozaki, Miyako Yoshioka,
Kentaro Masujin, Morikazu Imamura, Yoshifumi Iwamaru, Yuichi Matsuura, Kohtaro
Miyazawa, Shigeo Fukuda, Takashi Yokoyama, and Shirou Mohri
Author affiliations: Author affiliations: National Agriculture and Food
Research Organization, Tsukuba, Japan (H. Okada, Y. Murayama, N. Shimozaki, M.
Yoshioka, K. Masujin, M. Imamura, Y. Iwamaru, Y. Matsuura, K. Miyazawa, T.
Yokoyama, S. Mohri); Hokkaido Research Organization, Shintoku, Japan (S. Fukuda)
Acknowledgment
This work was supported by a grant-in-aid from the BSE and Other Prion
Disease Project of the Ministry of Agriculture, Forestry and Fisheries, Japan.
References
1.Murayama Y, Yoshioka M, Masujin K, Okada H, Iwamaru Y, Imamura M,
Sulfated dextrans enhance in vitro amplification of bovine spongiform
encephalopathy PrPSc and enable ultrasensitive detection of bovine PrPSc. PLoS
ONE. 2010;5:e13152. DOIPubMed 2.Okada H, Iwamaru Y, Imamura M, Masujin K,
Matsuura Y, Murayama Y, Detection of disease-associated prion protein in the
posterior portion of the small intestine involving the continuous Peyer’s patch
in cattle orally infected with bovine spongiform encephalopathy agent.
Transbound Emerg Dis. 2011;58:333–43. DOIPubMed 3.Haley NJ, Seelig DM, Zabel MD,
Telling GC, Hoover EA. Detection of CWD prions in urine and saliva of deer by
transgenic mouse bioassay. PLoS ONE. 2009;4:e4848. DOIPubMed 4.Tamgüney G, Richt
JA, Hamir AN, Greenlee JJ, Miller MW, Wolfe LL, Salivary prions in sheep and
deer. Prion. 2012;6:52–61. DOIPubMed 5.Vascellari M, Nonno R, Mutinelli F,
Bigolaro M, Di Bari MA, Melchiotti E, PrPSc in salivary glands of
scrapie-affected sheep. J Virol. 2007;81:4872–6. DOIPubMed 6.Maddison BC, Rees
HC, Baker CA, Taema M, Bellworthy SJ, Thorne L, Prions are secreted into the
oral cavity in sheep with preclinical scrapie. J Infect Dis. 2010;201:1672–6.
DOIPubMed 7.Gough KC, Baker CA, Rees HC, Terry LA, Spiropoulos J, Thorne L, The
oral secretion of infectious scrapie prions occurs in preclinical sheep with a
range of PRNP genotypes. J Virol. 2012;86:566–71. DOIPubMed 8.Mathiason CK,
Powers JG, Dahmes SJ, Osborn DA, Miller KV, Warren RJ, Infectious prions in the
saliva and blood of deer with chronic wasting disease. Science. 2006;314:133–6.
DOIPubMed 9.Brown P, Andréoletti O, Bradley R, Budka H, Deslys JP, Groschup M,
WHO tables on tissue infectivity distribution in transmissible spongiform
encephalopathies. Geneva: World Health Organization; 2010 [cited 2011 Nov 2]. http://www.who.int/bloodproducts/tablestissueinfectivity.pdf
Figure Figure. . Western blot detection, using the serial protein
misfolding cyclic amplification technique, of the abnormal (disease-associated)
form of the prion protein (PrPSc) in concentrated saliva samples from 3 cows
experimentally...
Suggested citation for this article: Okada H, Murayama Y, Shimozaki N,
Yoshioka M, Masujin K, Imamura M, et al. Prion in saliva of bovine spongiform
encephalopathy–infected cattle [letter]. Emerg Infect Dis [Internet]. 2012 Dec
[date cited]. DOI: http://dx.doi.org/10.3201/eid1812.120528
DOI: 10.3201/eid1812.120528
DISSERTATION
CHRONIC WASTING DISEASE: A MODEL FOR PRION TRANSMISSION VIA SALIVA AND
URINE
Submitted by Nicholas James Haley
Department of Microbiology, Immunology and Pathology In partial fulfillment
of the requirements For the Degree of Doctor of Philosophy Colorado State
University Fort Collins, Colorado Summer 2010
ABSTRACT OF DISSERTATION CHRONIC WASTING DISEASE: A MODEL FOR PRION
TRANSMISSION VIA SALIVA AND URINE
Chronic wasting disease (CWD) of cervids is a prion disease distinguished
by its high level of transmissibility, wherein bodily fluids and excretions are
thought to play an important role. Typical of all prion diseases, CWD is
characterized by the forced conversion of the normal prion protein (PrPC) into a
misfolded isoform (PrPCWD), which has been shown to accumulate primarily in
tissues of the lymphoid and nervous systems, though has also been found in other
peripheral tissues including elements of the cardiovascular, musculoskeletal,
and urogenital systems. Despite strong evidence that natural infection is
acquired from the environment, as well as saliva and blood, a more thorough
evaluation of excreta, including saliva, urine, and feces, is essential for a
comprehensive foundation for (1) understanding how environmental
CWDcontamination occurs, (2) developing in vitro assays for the antemortem
identification of CWD-infected cervids, and (3) demonstrating the pathogenesis
of the disease in the natural host.
In this dissertation, two approaches are used to identify infectious CWD
prions and PrPCWD in the bodily fluids and tissues of CWD-exposed white-tailed
deer: a novel bioassay system using a transgenic mouse line expressing the
cervid PrP protein (Tg[CerPrP] mice), and a recently developed prion
amplification assay known as serial iv protein misfolding cyclic amplification
(sPMCA). In conjunction with immunohistochemistry and western blotting, these
two assays were used to definitively identify CWD prions in saliva and urine, in
addition to elements of the lymphoreticular system, central and peripheral
nervous systems, and urogenital and oropharyngeal tissues. In initial
experiments, concentrated urine and saliva samples from terminal CWD+
white-tailed deer, suspected of harboring infectious CWD prions, was assessed by
Tg[CerPrP] bioassay and sPMCA. Authentic prion infectivity was detected in urine
and saliva using both detection systems in the case of urine, though only mouse
bioassay successfully demonstrated CWD prions in saliva. The concentration of
abnormal prion protein in bodily fluids was very low, as indicated by:
undetectable PrPCWD levels by traditional assays (western blot, ELISA) and
prolonged incubation periods and incomplete TSE attack rates in inoculated
Tg[CerPrP] mice. These findings helped to extend the understanding of CWD prion
shedding and transmission and portend the detection of infectious prions in body
fluids in other prion infections.
Based on the identification of CWD prions in saliva (“prionsialia”) and
urine (“prionuria”), I next sought to determine whether deer previously exposed
orally to urine and feces from CWD+ sources, while conventional test-negative,
may actually be harboring very low level CWD infection not evident in the 19
month observation period in initial cervid bioassay studies. A selection of
tissues, including those of the lymphoreticular and both central and peripheral
nervous systems were fully examined, initially using Tg[CerPrP] bioassay to
demonstrate true infectivity, and secondarily with sPMCA. Positive controls
consisted of issues from CWD+ deer exposed orally to saliva; negative control
tissue sets were collected from deer exposed orally and intracranially to
v
CWD-negative brain. PrPCWD was detected in the tissues of orally exposed
deer by both sPMCA and Tg[CerPrP] mouse bioassay; each assay revealed very low
levels of CWD prions previously undetectable by western blot, ELISA, or IHC.
Serial PMCA analysis of individual tissues identified that obex alone was
positive in urine/feces exposed deer. PrPCWD was amplified from both LRS and
neural tissues of positive control deer but not from the same tissues of
negative control deer. Detection of subclinical infection in deer orally exposed
to urine and feces (1) suggests that a prolonged subclinical state can exist
such that observation periods in excess of two years may be needed to detect CWD
infection, and (2) illustrates the sensitive and specific application of sPMCA
in the diagnosis of low-level prion infection.
Despite the confirmation of infectious prions in urine and saliva, along
with conventional test-negative deer exposed to urine and feces, the manner in
which infectivity is transferred to these excreta is unknown. To address this, I
went on to apply sPMCA to tissues associated with production and excretion of
urine and saliva in an effort to seek proximal sources of prion shedding. I
blindly analyzed oropharyngeal and urogenital tissues, reproducibly
demonstrating PrPCWD in each tissue examined in 3 rounds of sPMCA; whereas blood
samples from the same animals and concurrent negative controls remained
negative. Tissue distribution was affected by route of inoculation and CNS
burden. The identification of PrPCWD in bodily fluids and conventional-test
negative tissues – in the absence of detection by conventional methods – may
indicate the presence of protease-sensitive infectious prions in excretory
tissues not revealed by assays employing PK digestion or other means to remove
PrPC reactivity.
vi
The continued evaluation of bodily fluids and peripheral tissues via sPMCA
may therefore allow additional insights into prion transmission, trafficking,
and pathogenesis.
Nicholas James Haley Department of Microbiology, Immunology and Pathology
Colorado State University Fort Collins, CO 80523 Summer 2010
snip...
In summary, this study demonstrates for the first time amplifiable PrPCWD
in various organs and tissues associated with prionsialia and prionuria. The
ultimate source and mechanism of release into bodily fluids remain unknown,
though elevated levels in both salivary gland and urinary bladder provides
strong evidence that these tissues play a crucial role in prion excretion. In
addition, the source and route of inoculation weighed heavily on the terminal
peripheral distribution of PrPCWD, as did an individual’s apparent central
nervous system burden. Finally, while this discovery provides evidence for prion
invasion of peripheral excretory tissues, the timing of infiltration during CWD
infection and the protease resistance profile of these prions warrant future
studies in serial pathogenesis and detection of alternate infectious prion
species.
Sunday, July 03, 2011
Prion Disease Detection, PMCA Kinetics, and IgG in Urine from
Naturally/Experimentally Infected Scrapie Sheep and Preclinical/Clinical CWD
Deer
Thursday, June 09, 2011
Detection of CWD prions in salivary, urinary, and intestinal tissues of
deer: potential mechanisms of prion shedding and transmission
CHRONIC WASTING DISEASE: A MODEL FOR PRION TRANSMISSION VIA SALIVA AND
URINE
Sunday, December 06, 2009
Detection of Sub-Clinical CWD Infection in Conventional Test-Negative Deer
Long after Oral Exposure to Urine and Feces from CWD+ Deer
Wednesday, March 18, 2009
Detection of CWD Prions in Urine and Saliva of Deer by Transgenic Mouse
Bioassay
*** Tuesday, September 02, 2008
Detection of infectious prions in urine (Soto et al Available online 13
August 2008.)
Friday, October 26, 2012
CHRONIC WASTING DISEASE CWD PENNSYLVANIA GAME FARMS, URINE ATTRACTANT
PRODUCTS, BAITING, AND MINERAL LICKS
TSS
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