Monday, November 19, 2012

Prion in Saliva of Bovine Spongiform Encephalopathy–Infected Cattle


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. . 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)


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.


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].

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:

DOI: 10.3201/eid1812.120528



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


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


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.


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


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


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



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