Sunday, February 11, 2018

Real-Time Quaking-Induced Conversion Detection of Bovine Spongiform Encephalopathy Prions in a Subclinical Steer


Front. Vet. Sci., 19 January 2018 |

Real-Time Quaking-Induced Conversion Detection of Bovine Spongiform Encephalopathy Prions in a Subclinical Steer

imageSoyoun Hwang1, imageM. Heather West Greenlee2, imageAnne Balkema-Buschmann3, imageMartin H. Groschup3, imageEric M. Nicholson1 and imageJustin J. Greenlee1*

1United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Virus and Prion Research Unit, Ames, IA, United States

2Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, United States

3Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald, Germany

Bovine spongiform encephalopathy (BSE) belongs to a group of fatal prion diseases that result from the misfolding of the cellular prion protein (PrPC) into a pathogenic form (PrPSc) that accumulates in the brain. In vitro assays such as serial protein misfolding amplification and real-time quaking-induced conversion (RT-QuIC) allow assessment of the conversion of PrPC to PrPSc. RT-QuIC can be used for the detection of prions in a variety of biological tissues from humans and animals. However, there is no such comparison of RT-QuIC data between BSE positive and presymptomatic cattle. Further, the current study assesses prion distribution in multiple brain regions of clinically ill or subclinical animals. Here, we compare RT-QuIC reactions seeded with brain samples collected from experimentally inoculated cattle that were clinically ill or subclinically affected with BSE. The results demonstrate RT-QuIC seeding in various brain regions of an animal with subclinical BSE despite being determined negative by immunohistochemistry. Bioassay of the subclinical animal and RT-QuIC of brainstem from inoculated knockout (PRNP−/−) cattle were used to confirm infectivity in the subclinical animal and determine that RT-QuIC reactions were not the result of residual inoculum, respectively. These results confirm that RT-QuIC is a highly sensitive prion detection assay that can detect prions in a steer prior to the onset of clinical signs of BSE.


Discussion and Conclusion With this study, we provide proof of concept that RT-QuIC is a highly useful method for the detection of BSE prions in the brains of cattle with subclinical BSE. In the current study, an animal inoculated with classical BSE (No. 1) did not develop clinical illness and tested negative by standard diagnostics performed on brainstem despite a prolonged observation period of 31 months. When brainstem tested negative by EIA, we tested additional brain regions by EIA and RT-QuIC and compared the results to those of animals clinically ill with BSE in an effort to explain the failure of this animal to develop clinical signs. Prion distribution differences between subclinical and clinical animals led us to perform additional RT-QuIC reactions in PRNP knockout animals to ensure our RT-QuIC results were not due to the presence of residual TSE inoculum. Prions were not detected in brain samples from PRNP knockout animals by WB, EIA, IHC, or RT-QuIC.

In the current study, RT-QuIC detected lower levels of prions than traditional diagnostic tools. IHC and WB failed to identify any PrPSc in the subclinical animal (No. 1), while testing multiple brain regions by EIA and RT-QuIC allowed us to detect misfolded protein in midbrain and thalamus. Further, we were able to detect seeding activity in brain regions including cerebrum and cerebellum that were negative by EIA. This result is consistent with a recent study that demonstrated that RT-QuIC is more sensitive for the detection of PrPCWD during early infection than tyramide signal amplification-IHC (35).

Real-time quaking-induced conversion has been successfully used for sensitive and specific detection of prion diseases for humans and animals (11–13, 15–17, 36). We previously reported different types of BSE prions can be detected and differentiated using RT-QuIC with recombinant bovine prion protein (29), and the previous work of others evaluated detection and discrimination of classical, H-, and L-BSE prions by full-length chimeric hamster-sheep prion proteins, N-terminally truncated hamster prion protein [90–231] (37), full-length sheep protein, and full-length bank vole protein (a.a. 23–230). There has been significant effort to detect prions in presymptomatic diagnostic samples including CSF, blood, urine, saliva, and nasal brushings by RT-QuIC [7–16]. CWD prions were detected in urine collected from presymptomatic deer and in fecal extracts by using RT-QuIC (38) suggesting potential applications in CWD surveillance and control.

In the present study, there were differences in prion distribution between the brains of subclinical and clinically affected cattle after intracranial BSE inoculation. In the subclinically affected steer (No. 1), the highest levels of prions accumulated in the thalamus and midbrain, while no prions were detected in the brainstem. Most likely this reflects prion accumulation in the location that the inoculum was deposited without further spread into other brain regions. A study comparing preclinical cattle infected naturally with BSE to clinically affected cattle either naturally or experimentally infected with BSE by the oral route found the most abundant PrPSc in the brainstem area (39), which is consistent with ascension to the brain from the gut by sympathetic and parasympathetic projections (40). In our experiment, abundant prions were observed in the brainstem of cattle with clinical signs of BSE, which is similar to the amount in their thalamus or midbrain regions. Interestingly, prions in the brainstem of cattle with clinical evidence of BSE seeded the RT-QuIC reactions faster than any other brain region despite the brainstem area having lower EIA OD values (Table 2) in comparison to other brain regions. This suggests that higher concentrations of prions do not necessarily seed the reaction faster. Perhaps prions of the brainstem exist in a preferred conformation for better conversion despite being present in lower concentrations.

Bioassay of brainstem from steer No. 1 confirmed infectivity and RT-QuIC results despite being negative by IHC, WB, and EIA. Bioassay was performed with a 10% homogenate as compared to 1% for the original classical BSE inoculum, yet the average incubation period longer confirming lower infectivity in the brainstem of the subclinical steer. We previously reported that the retinas of animals inoculated with the agent of classical BSE become substantially thinner than both their baseline (preinoculation) thickness and the thickness of sham-inoculated control animals (22). This decrease in retinal thickness preceded the onset of clinical signs by an average of 10 months. It is interesting to note that in the previous study steer No. 1 had a substantially decreased retinal thickness value at 12 months postinoculation and would have been classified as “positive” by these previously reported criteria (22).

After being observed for nearly 9 months longer than other cattle in the same experimental group, a steer intracranially inoculated with classical BSE was euthanized without demonstrating clinical signs suggestive of prion disease. WB and IHC failed to demonstrate PrPSc in any region of the brain. In an effort to investigate the reason that this animal failed to develop clinical signs, further studies were conducted using RT-QuIC, which demonstrated high levels of prions in the thalamus and midbrain. In summary, we demonstrate that RT-QuIC can be useful to detect prions in cattle with subclinical BSE and that BSE prions can be differently distributed in the brain regions as incubation progresses.


BSE - ATYPICAL LESION DISTRIBUTION (RBSE 92-21367) statutory (obex only) diagnostic criteria CVL 1992

Wednesday, July 15, 2015

Additional BSE TSE prion testing detects pathologic lesion in unusual brain location and PrPsc by PMCA only, how many cases have we missed?

***however in 1 C-type challenged animal, Prion 2015 Poster Abstracts S67 PrPsc was not detected using rapid tests for BSE.

***Subsequent testing resulted in the detection of pathologic lesion in unusual brain location and PrPsc detection by PMCA only.

*** IBNC Tauopathy or TSE Prion disease, it appears, no one is sure ***

Posted by Terry S. Singeltary Sr. on 03 Jul 2015 at 16:53 GMT

Discussion: The C, L and H type BSE cases in Canada exhibit molecular characteristics similar to those described for classical and atypical BSE cases from Europe and Japan.

*** This supports the theory that the importation of BSE contaminated feedstuff is the source of C-type BSE in Canada.

*** It also suggests a similar cause or source for atypical BSE in these countries. ***

see page 176 of 201 pages...tss

*** Singeltary reply ; Molecular, Biochemical and Genetic Characteristics of BSE in Canada Singeltary reply;


Experimental sheep BSE prions generate the vCJD phenotype when serially passaged in transgenic mice expressing human prion protein

Detection of PrPBSE and prion infectivity in the ileal Peyer’s patch of young calves as early as 2 months after oral challenge with classical bovine spongiform encephalopathy

Ivett Ackermann, Anne Balkema-Buschmann, Reiner Ulrich, Kerstin Tauscher, James C. Shawulu, Markus Keller, Olanrewaju I. Fatola, Paul Brown and Martin H. GroschupEmail authorView ORCID ID profile

Veterinary Research201748:88

Received: 22 August 2017Accepted: 1 December 2017Published: 19 December 2017


In classical bovine spongiform encephalopathy (C-BSE), an orally acquired prion disease of cattle, the ileal Peyer’s patch (IPP) represents the main entry port for the BSE agent. In earlier C-BSE pathogenesis studies, cattle at 4–6 months of age were orally challenged, while there are strong indications that the risk of infection is highest in young animals. In the present study, unweaned calves aged 4–6 weeks were orally challenged to determine the earliest time point at which newly formed PrPBSE and BSE infectivity are detectable in the IPP. For this purpose, calves were culled 1 week as well as 2, 4, 6 and 8 months post-infection (mpi) and IPPs were examined for BSE infectivity using a bovine PrP transgenic mouse bioassay, and for PrPBSE by immunohistochemistry (IHC) and protein misfolding cyclic amplification (PMCA) assays. For the first time, BSE prions were detected in the IPP as early as 2 mpi by transgenic mouse bioassay and PMCA and 4 mpi by IHC in the follicular dendritic cells (FDCs) of the IPP follicles. These data indicate that BSE prions propagate in the IPP of unweaned calves within 2 months of oral uptake of the agent.


In summary, our study demonstrates for the first time PrPBSE (by PMCA) and prion infectivity (by mouse bioassay) in the ileal Peyer’s patch (IPP) of young calves as early as 2 months after infection. From 4 mpi nearly all calves showed PrPBSE positive IPP follicles (by IHC), even with PrPBSE accumulation detectable in FDCs in some animals. Finally, our results confirm the IPP as the early port of entry for the BSE agent and a site of initial propagation of PrPBSE and infectivity during the early pathogenesis of the disease. Therefore, our study supports the recommendation to remove the last four metres of the small intestine (distal ileum) at slaughter, as designated by current legal requirements for countries with a controlled BSE risk status, as an essential measure for consumer and public health protection.


APHIS USDA Food Safety Research Priorities and the one you missed, the coming storm BSE CWD Scrapie TSE Prion


Detection of PrPBSE and prion infectivity in the ileal Peyer’s patch of young calves as early as 2 months after oral challenge with classical bovine spongiform encephalopathy

Thursday, November 16, 2017 

Texas Natural Meats Recalls Beef Products Due To Possible Specified Risk Materials Contamination



Bovine Spongiform Encephalopathy BSE TSE Prion (aka mad cow disease) Report December 14, 2017 2017


Chronic Wasting Disease CWD TSE Prion (aka mad deer disease) Update USA December 14, 2017


Canada CFIA updating its national CWD TSE PRION efforts to eradicate disease farmed cervid NOT successful December 14, 2017




Creutzfeldt Jakob Disease CJD National Prion Disease Pathology Surveillance Center Cases Examined to December 14, 2017

Tuesday, December 12, 2017 

Neuropathology of iatrogenic Creutzfeldt–Jakob disease and immunoassay of French cadaver-sourced growth hormone batches suggest possible transmission of tauopathy and long incubation periods for the transmission of Abeta pathology


Chronic wasting disease management in ranched elk using rectal biopsy testing Research Paper 09 Feb 2018

Back around 2000, 2001, or so, I was corresponding with officials abroad during the bse inquiry, passing info back and forth, and some officials from here inside USDA aphis FSIS et al. In fact helped me get into the USA 50 state emergency BSE conference call way back. That one was a doozy. But I always remember what “deep throat” I never knew who they were, but I never forgot;

Some unofficial information from a source on the inside looking out -


As early as 1992-3 there had been long studies conducted on small pastures containing scrapie infected sheep at the sheep research station associated with the Neuropathogenesis Unit in Edinburgh, Scotland. Whether these are documented...I don't know. But personal recounts both heard and recorded in a daily journal indicate that leaving the pastures free and replacing the topsoil completely at least 2 feet of thickness each year for SEVEN years....and then when very clean (proven scrapie free) sheep were placed on these small pastures.... the new sheep also broke out with scrapie and passed it to offspring. I am not sure that TSE contaminated ground could ever be free of the agent!! A very frightening revelation!!!

---end personal email---end...tss


Assessment of Chronic Wasting Disease Prion Shedding in Deer Saliva with Occupancy Modeling


Dehydration of prions on environmentally relevant surfaces protects them from inactivation by freezing and thawing




Creutzfeldt Jakob Disease CJD National Prion Disease Pathology Surveillance Center Cases Examined to December 14, 2017

Tuesday, December 12, 2017 

Neuropathology of iatrogenic Creutzfeldt–Jakob disease and immunoassay of French cadaver-sourced growth hormone batches suggest possible transmission of tauopathy and long incubation periods for the transmission of Abeta pathology


Creutzfeldt Jakob Disease United States of America USA and United Kingdom UK Increasing and Zoonotic Pontential From Different Species


*** Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States revisited 2017

Singeltary et al


Variably protease-sensitive prionopathy: A new sporadic disease of the prion protein 

Here we go folks. AS predicted. THIS JUST OUT !

Saturday, June 13, 2009

Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States 2003 revisited 2009 

Sunday, August 09, 2009

CJD...Straight talk with...James Ironside...and...Terry Singeltary... 2009 

FRIDAY, AUGUST 11, 2017 

Infectivity in bone marrow from sporadic CJD patients

Bioassays in transgenic mice expressing the human prion protein revealed the presence of unexpectedly high levels of infectivity in the bone marrow from seven out of eight sCJD cases. These findings may explain the presence of blood-borne infectivity in sCJD patients. They also suggest that the distribution of prion infectivity in peripheral tissues in sCJD patients could be wider than currently believed, with potential implications for the iatrogenic transmission risk of this disease. 

*** Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery *** 

Gibbs CJ Jr, Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC. Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892. 

Stereotactic multicontact electrodes used to probe the cerebral cortex of a middle aged woman with progressive dementia were previously implicated in the accidental transmission of Creutzfeldt-Jakob disease (CJD) to two younger patients. The diagnoses of CJD have been confirmed for all three cases. More than two years after their last use in humans, after three cleanings and repeated sterilisation in ethanol and formaldehyde vapour, the electrodes were implanted in the cortex of a chimpanzee. Eighteen months later the animal became ill with CJD. This finding serves to re-emphasise the potential danger posed by reuse of instruments contaminated with the agents of spongiform encephalopathies, even after scrupulous attempts to clean them. 


*** Minimise transmission risk of CJD and vCJD in healthcare settings Updated 10 August 2017

Diagnosis and Reporting of Creutzfeldt-Jakob Disease 
Singeltary, Sr et al. JAMA.2001; 285: 733-734. Vol. 285 No. 6, February 14, 2001 JAMA Diagnosis and Reporting of Creutzfeldt-Jakob Disease 

To the Editor: 

In their Research Letter, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally. 

Terry S. Singeltary, Sr Bacliff, Tex 

1. Gibbons RV, Holman RC, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease in the United States: 1979-1998. JAMA. 2000;284:2322-2323. 

Terry S. Singeltary Sr.