Prion in saliva of bovine spongiform encephalopathy-infected cattle.
Article Type: Letter to the editor
Subject: Medical research
Medicine, Experimental
Authors: Okada, Hiroyuki
Murayama, Yuichi
Shimozaki, Noriko
Yoshioka, Miyako
Masujin, Kentaro
Imamura, Morikazu
Iwamaru, Yoshifumi
Matsuura, Yuichi
Miyazawa, Kohtaro
Fukuda, Shigeo
Yokoyama, Takashi
Mohri, Shirou
Pub Date: 12/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: Dec, 2012 Source Volume: 18 Source Issue: 12
Product: Product Code: 8000200 Medical Research; 9105220 Health Research Programs; 8000240 Epilepsy & Muscle Disease R&D NAICS Code: 54171 Research and Development in the Physical, Engineering, and Life Sciences; 92312 Administration of Public Health Programs
Accession Number: 313345675
Full Text: 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 ([PrP.sup.Sc]) 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 [PrP.sup.Sc] 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 [PrP.sup.Sc] 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 [PrP.sup.Sc].

We collected saliva samples from animals at 4 monthly intervals, beginning in 2009, 56 months after inoculation. Samples were stored at -80[degrees]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 [PrP.sup.Sc] in brains collected at necropsy from the 3 cows examined. In addition, using the sPMCA technique, we detected [PrP.sup.Sc] 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.

After saliva samples underwent 3 rounds of amplification, we detected [PrP.sup.Sc] 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 [PrP.sup.Sc] at round 4 of amplification increased as the disease progressed (Figure). Because [PrP.sup.Sc] signal could be detected in BSE-infected brain homogenates diluted up to 10-10 after 2 rounds of amplification (1), we estimated [PrP.sup.Sc] levels in the nonconcentrated original saliva samples to be lower than those in BSE-infected brain homogenate diluted to 10-12. No [PrP.sup.Sc] signal was detected in samples collected from the 3 cows 3-5 months before the onset of clinical symptoms or from agematched noninfected controls, even after 4 rounds of amplification.

We demonstrated the presence of [PrP.sup.Sc] 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 [PrP.sup.Sc] is likely to be detected in the saliva of BSE-affected cattle during the clinical stage of disease, after accumulation of [PrP.sup.Sc] in the brain. [PrP.sup.Sc] 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 [PrP.sup.Sc] 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 scrapieaffected sheep (4); the immunolabeled [PrP.sup.Sc] accumulated in the salivary glands of scrapie-affected sheep (5). A low level of [PrP.sup.Sc] was detected in concentrated buccal swab samples of preclinical scrapie-infected sheep by using sPMCA (6,7). These results suggest that small amounts of [PrP.sup.Sc] 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.

This work was supported by a grantin-aid from the BSE and Other Prion Disease Project of the Ministry of Agriculture, Forestry and Fisheries, Japan.

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: 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); and Hokkaido Research Organization, Shintoku, Japan (S. Fukuda)

DOI: 10.3201/eid1812.120528


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Address for correspondence: Yuichi Murayama, Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan; email:
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