Friday, February 08, 2019

Prion protein polymorphisms associated with reduced CWD susceptibility limit peripheral PrPCWD deposition in orally infected white-tailed deer

Prion protein polymorphisms associated with reduced CWD susceptibility limit peripheral PrPCWD deposition in orally infected white-tailed deer

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BMC Veterinary Research201915:50
  • Received: 1 October 2018
  • Accepted: 22 January 2019
  • Published: 

Abstract

Background

Chronic wasting disease (CWD) is a prion disease affecting members of the Cervidae family. PrPC primary structures play a key role in CWD susceptibility resulting in extended incubation periods and regulating the propagation of CWD strains. We analyzed the distribution of abnormal prion protein (PrPCWD) aggregates in brain and peripheral organs from orally inoculated white-tailed deer expressing four different PRNP genotypes: Q95G96/Q95G96 (wt/wt), S96/wt, H95/wt and H95/S96 to determine if there are substantial differences in the deposition pattern of PrPCWD between different PRNP genotypes.

Results

Although we detected differences in certain brain areas, globally, the different genotypes showed similar PrPCWD deposition patterns in the brain. However, we found that clinically affected deer expressing H95 PrPC, despite having the longest survival periods, presented less PrPCWD immunoreactivity in particular peripheral organs. In addition, no PrPCWD was detected in skeletal muscle of any of the deer.

Conclusions

Our data suggest that expression of H95-PrPC limits peripheral accumulation of PrPCWD as detected by immunohistochemistry. Conversely, infected S96/wt and wt/wt deer presented with similar PrPCWD peripheral distribution at terminal stage of disease, suggesting that the S96-PrPC allele, although delaying CWD progression, does not completely limit the peripheral accumulation of the infectious agent.

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PrPCWD deposition in kidney

Kidney samples were evaluated from 9 of the 10 CWD clinical deer. Positive immunolabeling was found in all evaluated kidney samples from wt/wt and S96/wt deer. PrPCWD staining was consistently associated with arterial vessels, showing a periarterial and periarteriolar deposition. The most abundant immunolabeling was detected in the wall of the main renal artery and arcuate arteries, which locate at the junction of the renal cortex and the renal medulla and arise from interlobar arteries. Interestingly, no PrPCWD deposits were found in any of the evaluated kidney samples from the H95/wt and the H95/S96 deer (Table 2, Fig. 6). In addition to the PrPCWD aggregates observed in arterial vessels, one wt/wt deer had strong PrPCWD deposition associated with foci of inflammatory cells (D1) affecting renal glomeruli, which was compatible with a moderate interstitial glomerulonephritis (Fig. 7a).

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PrPCWD deposition in salivary glands

PrPCWD deposits found in salivary glands were mild and scattered. Positive immunolabeling was found in the interstitial tissue between acini, whereas no intracellular deposits were detected in acinar cells or within salivary ducts in any of the samples evaluated. Parasympathetic ganglia neurons innervating the salivary gland tissue from a wt/wt deer (D4), presented strong intraneuronal immunolabeling (Fig. 6c). IHC deposits were observed in parotid and submandibular salivary glands, whereas all the evaluated sublingual glands samples were negative for PrPCWD immunostaining. Deposition in the interstitial tissue was especially evident in the submandibular salivary gland of D7 (Fig. 6d). Positive immunolabeling was detected in at least one salivary gland sample from all clinically affected deer with the exception of H95/wt and H95/S96 deer, which showed no PrPCWD deposits in any of the salivary glands evaluated.

PrPCWD deposition in other organs

PrPCWD immunolabeling was detected in the lungs of D1. This deer showed mild lung interstitial inflammation and edema. PrPCWD aggregates were observed to be associated with inflammatory cell foci and in the cells of the bronchiolar epithelium (Fig. 7b). This deer also showed, as mentioned previously, positive immunolabeling related to the accumulation of inflammatory cells in the kidney (Fig. 7a). Eye samples were collected from all deer included in this study. In addition to the positive immunolabeling of the optic nerve, described above, all deer were also positive for PrPCWD IHC deposition in the retina. All collected liver samples from the clinically affected deer were IHC negative (Table 2).
Corresponding negative tissue controls from uninfected deer were included when available. These tissues did not present any immunolabeling, confirming the specificity of the immunohistochemical detection (Tables 1 and 2).

Discussion

We found that the intensity and distribution of PrPCWD deposits in brain and peripheral tissues of PRNP polymorphic (i.e. different PrPC primary structures) white-tailed deer was distinct from Q95G95 (wt) homozygous deer exposed to the same prion strain (i.e. Wisc-1). We have previously shown that H95 and S96 PRNPpolymorphisms play a key role in CWD susceptibility, increasing survival periods and having dramatic effects on the propagation of CWD strains [161718192037].
Our results show that deer expressing the H95-PrPC presented a more limited peripheral distribution of PrPCWD compared to wt/wt and S96/wt deer. Under identical experimental conditions and disease stage, the number of organs with positive immunolabeling was reduced in deer with H95-PrPC allelotypes [18]. The most significant differences in PrPCWD deposition between deer with different PRNP genotypes were found in pancreas, heart, kidney and intestine samples. Both deer expressing the H95-PrPC showed no immunolabeling or reduced accumulation of PrPCWD aggregates in these tissues.
The presence of PrPCWD in endocrine tissues has been previously described in the adrenal medulla, the pituitary gland and islets of Langerhans in the pancreas of CWD-affected cervids [3132], results that agree with our observations in CWD-infected wt/wt and S96/wt deer. Only deer of these genotypes (Fig. 3ab) showed moderate to strong chromagen deposition in islets of Langerhans, which are innervated by the vagus nerve [32]. Differences in adrenal glands immunolabeling were minor between deer expressing different PrPCpolymorphisms. Consistent with wt-PrPC being the cognate substrate for Wisc-1 homologous prion conversion, wt/wt deer presented higher and widespread PrPCWD deposition compared to deer of other genotypes.
The distribution of PrPCWD aggregates was also limited in hearts of animals expressing the H95-PrPC allelotype. Cardiac tissues were collected from D6 (S96/wt), D9 (H95/wt) and D10 (H95/S96). PrPCWD accumulation was detected in multiple heart samples from D6, affecting separated groups of cardiac myocytes (Fig. 4a). Conversely, no immunolabeling was observed in any heart sample from deer expressing the H95-PrPC (Fig. 4bc).
A distinct pattern of distribution of PrPCWD was also observed in intestinal tissues for H95 carriers. It is not surprising that strong immunolabeling was observed in gut-associated lymphoid and nervous tissues as these are known to be the first sites of PrPd accumulation following oral infection [26303138]. Nevertheless, H95/wt and H95/S96 deer, which had the longest incubation periods [18], showed more restricted or localized PrPCWD accumulation (Fig. 5). These findings however, do not necessarily indicate that these animals excrete a lower amount of prions or present a lack of infectivity in intestinal tissues.
PrPCWD deposits were observed in renal tissues of all wt/wt and S96/wt deer evaluated in the present study, whereas no immunopositive material was found in any of the kidney samples collected from deer expressing the H95 allele (Fig. 6). Immunohistochemical detection of PrPCWD in kidneys of CWD-infected white-tailed deer has previously only been reported in ectopic lymphoid follicles [3139]. PrPCWD in renal tissues has, however, been demonstrated by sPMCA [40] and it has been shown that CWD-infected cervids can shed infectious prions in urine [414243], although the proximal source of PrPCWD in urine is not known [40]. In the present study, PrPCWD positive immunolabeling of kidney samples was detected along the wall of the renal arteries, especially in the main renal artery and the wall of arcuate arteries (Fig. 6ab). In scrapie-affected sheep, prion deposition has been found in renal papillae and renal corpuscles [3344]. Periarterial and periarteriolar immunolabeling could be due to spread of prions through peripheral nerves [44] since the wall of these renal arteries is strongly innervated and sympathetic nerve fibers from the renal plexus enter the kidney accompanying the branches of the main renal artery. To our knowledge, this is the first description of PrPCWD deposition, detected by conventional techniques, in renal arteries of CWD-infected deer.
D1 also presented intense PrPCWD immunolabeling in the renal cortex associated with accumulations of inflammatory cells (Fig. 7a). Inflammatory processes affect prion pathogenesis and peripheral accumulation [4546], and chronic nephritis triggers prionuria in prion-infected mice [47]. In addition, PrPCWD shedding has been reported in CWD infected deer presenting with inflammatory kidney disease [41]. We cannot predict the effect of prion accumulation in the arterial walls on prionuria, however, we have observed that inflammatory kidney conditions greatly increase PrPCWD deposition in renal tissues from deer with CWD, which might increase shedding of PrPCWD within urine. This deer also showed PrPCWDaccumulation in the lungs associated with inflammatory cell foci and in the bronchiolar epithelium (Fig. 7b). PrP accumulation in the lung related to inflammatory conditions and in the epithelium of the bronchioles has been previously described in scrapie-affected sheep [3348]. Deer in our study were housed indoors with ample access to clean food and water [18]. It is likely that free-ranging deer would be at greater risk of coincident infections and commensurate inflammation that may influence the effect of protective alleles on susceptibility to CWD, tissue colonization by CWD prions and shedding.
In organs related to excreta production, differences in PrPCWD deposition were found in salivary glands between deer genotypes. PrPd in salivary glands, detected by conventional techniques, has been described in scrapie-affected sheep [49] and in the serous epithelial cells of the submandibular salivary gland in experimentally infected red deer [50]. Likewise, considerable PrPCWD amplifying activity, similar to that observed in brain, accumulates in salivary glands of cervids with CWD [40]. Comparison of single salivary gland sections identified PrPCWD immunostaining in wt/wt or S96/wt deer but not in deer expressing H95-PrPC (Table 2). Intense PrPCWD immunolabeling in ganglia cells immersed in the salivary gland tissue was observed in animal D4 (Fig. 6c).
Our observations suggest that deer expressing H95-PrPC have reduced centrifugal trafficking via descending nerves into peripheral tissues (i.e. salivary glands, pancreas, heart and kidney). This is consistent with previous observations in different experimental prion infections [3233364451]. However, it has been suggested that, in initial stages of CWD infection, PrPCWD may be trafficked via blood [26], and infectivity has been demonstrated in blood components [5253]. Therefore, we cannot exclude the hematogenous route as a complementary pathway of prion dissemination.
None of the clinically affected deer presented positive immunolabeling in any of the skeletal muscle samples evaluated, including the tongue. The absence of PrPdaccumulation in skeletal muscles detectable by IHC techniques has been reported in both naturally and experimentally prion infected deer [5455]. Nevertheless, although we did not detect PrPCWD deposition in skeletal muscle samples in this experimental Wisc-1 transmission to white-tailed deer, we cannot assume the complete absence of prion accumulation. Others have demonstrated the presence of PrPCWD in skeletal muscles by bioassay [56], Western Blot, PMCA and tissue-blotting [34].
The presence of prion deposits in skeletal muscles has previously been reported in neuromuscular spindles, which are highly innervated structures [3335] and, in CWD-infected white-tailed deer, in nerve fascicles [34]. Although most of the vagus nerve samples evaluated in the present study showed positive immunolabeling, sciatic nerve and brachial plexus samples presented sparse or no PrPCWD deposits (Table 2). Our results are similar to those in mule deer naturally infected with CWD [32]. We did not detect PrPCWD deposition in forelimb and hindlimb skeletal muscle samples, not even in the neuromuscular spindles. Due to the fact that brachial plexus and sciatic nerve innervate, respectively, the forelimb and hindlimb muscles, and taking into account that prions can spread to these muscles via these neural pathways [32], we can suggest that the scant or absent PrPCWD immunolabeling in brachial plexus and sciatic nerve samples from deer in our study may relate to the absence of deposits in these groups of muscles.
Cumulative evidence supports the limiting effect of H95 and S96 PrPCpolymorphisms on natural CWD infection and disease progression [16171837]. These PrPC allelic variants modulate CWD propagation and the efficiency of intraspecies CWD transmission [1957]. The passage of CWD prions in white-tailed deer expressing the H95-PrPC led to the emergence of the novel CWD strain H95+ [19]. This strain presented distinct biochemical and transmission properties, efficiently propagating in transgenic mice expressing deer S96-PrPC and in non-transgenic C57BL/6 mice [1920]. Wisc-1 propagation in deer expressing the H95-PrPC also presented limited peripheral PrPCWD accumulation.
As demonstrated in sheep with scrapie, PRNP genotype strongly influences the tropism and distribution of PrP deposits [232530]. In the present study, the observed effects of the H95 allele in prion distribution resemble those described for sheep expressing the resistance-associated allele ARR at codons 136, 154 and 171 of the prion protein. ARR/VRQ sheep, despite developing disease and accumulating PrPSc in the brain, present a much more limited and infrequent PrPSc distribution in lymphoid tissues compared to those with other susceptible genotypes [305859]. This effect is likely due to a modulation of the prion pathogenesis [2560] and it is not necessarily associated with the prolonged incubation period [25].
The similarities in peripheral PrPCWD accumulation between H95/wt and H95/S96 deer indicate a limiting role for the H95 amino acid substitution on the production and/or accumulation of PrPCWD. Similar observations have been made in goats expressing methionine at codon 142, which show reduced incidence of scrapie infection and a lower tendency to accumulate PrPSc outside the brain compared to 142 isoleucine homozygotes [5061]. In contrast, our findings suggest that H95-PrPC does not affect LRS involvement in CWD-affected deer (Table 1).
Influences of genotype on PrPCWD deposition pattern have been described in experimentally infected mule deer, with F225/S225 deer presenting with milder PrPCWD accumulation and limited tissue distribution compared to S225 homozygotes at identical intervals post-inoculation. This suggests the F225 amino acid variant limits prion conversion and delays PrPCWD tissue accumulation [3157]. Similar observations have been made in white-tailed deer expressing the S96 PrPC, which, compared to wt/wt deer, show reduced PrPCWD in brain and lymphoid tissues, consistent with slower disease progression [1726]. Deer of this genotype have also been reported to present lower PrPCWD immunostaining scores in the obex than wt/wt deer [6263]. However, observations were made in naturally infected animals in different stages of the disease. Although we observed certain differences between wt/wt and S96/wt deer in particular brain regions (Fig. 2), the overall intensity and peripheral distribution of PrPCWD was similar for both genotypes at the terminal stage of the disease (Tables 1 and 2). By contrast, the H95 polymorphism was a stronger driver of disease phenotype. The interference exerted by H95-PrPC in the replication and tissue accumulation of Wisc-1 prions relates to the biology of cervid PrPC polymorphisms and the evolution of CWD prion strains [1920].
Given that the diversity of CWD agents can be expanded in deer expressing PrPCpolymorphisms [192064], our findings cannot be generalized to include all potentially existing CWD strains which will likely have distinct host specific interactions. Prion disease characteristics, including the lesion distribution and the IHC phenotype of PrPd accumulation, are strongly influenced by the infecting prion strain [21656667]. Thus, it is possible that the reduced PrPCWDimmunolabeling observed in areas of the H95/S96 brain (Figs. 1 and 2) could be due to intra-species transmission barrier determined by the compatibility between the invading strain and the PrPC sequence of the host [6869]. Likewise, heterozygous deer presented lower levels of PrPCWD deposits in certain rostral brain areas, as compared to wt/wt deer (Fig. 2). These observations are consistent with the reduced amounts of PrPCWD as detected by Western blottimg [18]. Deer in the present study were orally inoculated with Wisc-1 prions [19], a CWD source obtained from animals homozygous for the wt-PrPC [18]. Therefore, transmission of the Wisc-1 strain into H95/S96 deer involves the adaptation of the infectious agent to this new host microenvironment, which could partially explain the reduced PrPCWD accumulation in particular brain regions, considering that the expression of wt-PrPC favors the propagation of the Wisc-1 strain [19].
The PrPCWD accumulated in the H95/S96 animal (H95-PrPCWD) [19] is more PK-sensitive than PrPCWD from deer with at least one wt allele [18]. Differences in PrP resistance to proteolytic degradation can lead to variable results in diagnostic tests. For example, atypical/Nor98 scrapie isolates are highly sensitive to PK digestion compared with classical scrapie strains, which leads to inconsistent diagnosis by PrPd IHC [70]. Therefore, this particular characteristic of H95-PrPCWDmay also explain the lower immunolabeling observed in the brain of H95/S96 deer. Nevertheless, prion disease neuropathological phenotypes, which include the PrPd profile, may depend on complex interactions between the infecting prion strain and host factors (e.g. the PRNP genotype) [23]. This host-pathogen interaction might explain the differences observed between deer genotypes with respect to the PrPCWD deposition in the cerebellum (Fig. 2). However, as mentioned, other CWD strains could differentially interact with PrP primary sequence and those effects should be further explored to understand how PrPCpolymorphisms modulate the propagation of CWD infectious agents.

Conclusions

The present study indicates that expression of the H95 PrPC polymorphism limits the intensity and distribution of PrPCWD aggregates in a wide variety of tissues and supports previous findings on the role deer PRNP genotype plays on the modulation of CWD progression and adaptation of CWD strains. Although breeding programs selecting for less susceptible PRNP genotypes can be effective in reducing scrapie prevalence in flocks [71], our data regarding the impact of deer PRNP genotypes need to be interpreted with caution. Animals expressing H95 PrPC, although presenting with a more limited prion distribution and longer incubation periods, represent the adaptation of a new strain [19]. Genetic enrichment for H95-PRNP alleles in deer herds may enhance the selection of H95+ or of novel strains with increased ability to propagate in genotypes including this PrPC polymorphism. The significantly longer incubation periods observed in deer with H95-PRNP alleles may not impact secretion of CWD (i.e., less CWD secreted over longer time periods). The emergence of new CWD strains could implicate a zoonotic potential [20].

Keywords

  • Prions
  • Prion diseases
  • Chronic wasting disease
  • CWD
  • PrPCWD
  • Peripheral tissues
  • Polymorphisms
  • Deer



for all you Michigan hunters still following cwd fake news expert on michigan-sportsman.com;

please be aware, michigan-sportsman.com fake news cwd expert and guru OTCARCHAR with new Keanu Reeves profile picture ;


Details image2.jpeg Tuesday at 12:07 PM#2 

 otcarcher

WE LOVE IT! Michigan Sportsman You've been here one season and counting!

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This statement from the link is not accurate.

"With an incubation period of up to two years"

Factually speaking, the incubation period can be 18 months to many years. Some genotypes prolong incubation times, some genotypes have shown to be immune to the disease, especially in certain Elk genotypes.

===

***> ''some genotypes have shown to be immune to the disease, especially in certain Elk genotypes.''

===

***at present, no cervid PrP allele conferring absolute resistance to prion infection has been identified. 

P-145 Estimating chronic wasting disease resistance in cervids using real time quaking- induced conversion 

Nicholas J Haley1, Rachel Rielinqer2, Kristen A Davenport3, W. David Walter4, Katherine I O'Rourke5, Gordon Mitchell6, Juergen A Richt2 1 Department of Microbiology and Immunology, Midwestern University, United States; 2Department of Diagnostic Medicine and Pathobiology, Kansas State University; 3Prion Research Center; Colorado State University; 4U.S. Geological Survey, Pennsylvania Cooperative Fish and Wildlife Research Unit; 5Agricultural Research Service, United States Department of Agriculture; 6Canadian Food Inspection Agency, National and OlE Reference Laboratory for Scrapie and CWD 

In mammalian species, the susceptibility to prion diseases is affected, in part, by the sequence of the host's prion protein (PrP). In sheep, a gradation from scrapie susceptible to resistant has been established both in vivo and in vitro based on the amino acids present at PrP positions 136, 154, and 171, which has led to global breeding programs to reduce the prevalence of scrapie in domestic sheep. In cervids, resistance is commonly characterized as a delayed progression of chronic wasting disease (CWD); at present, no cervid PrP allele conferring absolute resistance to prion infection has been identified. To model the susceptibility of various naturally-occurring and hypothetical cervid PrP alleles in vitro, we compared the amplification rates and efficiency of various CWD isolates in recombinant PrPC using real time quaking-induced conversion. We hypothesized that amplification metrics of these isolates in cervid PrP substrates would correlate to in vivo susceptibility - allowing susceptibility prediction for alleles found at 10 frequency in nature, and that there would be an additive effect of multiple resistant codons in hypothetical alleles. Our studies demonstrate that in vitro amplification metrics predict in vivo susceptibility, and that alleles with multiple codons, each influencing resistance independently, do not necessarily contribute additively to resistance. Importantly, we found that the white-tailed deer 226K substrate exhibited the slowest amplification rate among those evaluated, suggesting that further investigation of this allele and its resistance in vivo are warranted to determine if absolute resistance to CWD is possible. 

***at present, no cervid PrP allele conferring absolute resistance to prion infection has been identified. 

PRION 2016 CONFERENCE TOKYO 


''There are no known familial or genetic TSEs of animals, although polymorphisms in the PRNP gene of some species (sheep for example) may influence the length of the incubation period and occurrence of disease.'' 

c) The commonest form of CJD occurs as a sporadic disease, the cause of which is unknown, although genetic factors (particularly the codon 129 polymorphism in the prion protein gene (PRNP)) influence disease susceptibility. The familial forms of human TSEs (see Box 1) appear to have a solely genetic origin and are closely associated with mutations or insertions in the PRNP gene. Most, but not all, of the familial forms of human TSEs have been transmitted experimentally to animals. There are no known familial or genetic TSEs of animals, although polymorphisms in the PRNP gene of some species (sheep for example) may influence the length of the incubation period and occurrence of disease. 


the other part, these tissues and things in the body then shed or secrete prions which then are the route to other animals into the environment, so in particular, the things, the secretions that are infectious are salvia, feces, blood and urine. so pretty much anything that comes out of a deer is going to be infectious and potential for transmitting disease.

https://www.youtube.com/watch?v=bItnEElzuKo&index=6&list=PL7ZG8MkruQh3wI96XQ8_EymytO828rGxj

Texas Chronic Wasting Disease CWD TSE Prion Symposium 2018 posted January 2019 VIDEO SET 18 CLIPS

See Wisconsin update...terrible news, right after Texas updated map around 5 minute mark...


WISCONSIN CWD CAPTIVE CWD UPDATE VIDEO


cwd update on Wisconsin from Tammy Ryan...


Wyoming CWD Dr. Mary Wood

''first step is admitting you have a problem''

''Wyoming was behind the curve''

wyoming has a problem...


SATURDAY, JANUARY 19, 2019 

Texas Chronic Wasting Disease CWD TSE Prion Symposium 2018 posted January 2019 VIDEO SET 18 CLIPS


TUESDAY, JANUARY 29, 2019 

TEXAS REPORTS 2 MORE CWD TSE PRION ALL WILD CERVID TOTAL TO DATE 141



SATURDAY, JANUARY 19, 2019 

Texas Chronic Wasting Disease CWD TSE Prion Symposium 2018 posted January 2019 VIDEO SET 18 CLIPS


***> NORWAY CWD UPDATE December 2018

Report from the Norwegian Scientific Committee for Food and Environment (VKM) 2018: 16

Factors that can contribute to spread of CWD – an update on the situation in Nordfjella, Norway

Opinion of Panel on biological hazards of the Norwegian Scientific Committee for Food and Environment

13.12.2018

ISBN: 978-82-8259-316-8

ISSN: 2535-4019

Norwegian Scientific Committee for Food and Environment (VKM)

Po 222 Skøyen

0213 Oslo

Norway

FRIDAY, DECEMBER 14, 2018 

Norway, Nordfjella VKM 2018 16 Factors that can contribute to spread of CWD TSE Prion UPDATE December 14, 2018



THURSDAY, OCTOBER 25, 2018 

***> Norway New additional requirements for imports of hay and straw for animal feed from countries outside the EEA due to CWD TSE Prion


new link;

THURSDAY, OCTOBER 04, 2018 

Cervid to human prion transmission 5R01NS088604-04 Update



THURSDAY, FEBRUARY 07, 2019 

CWD TSE Prion, and Processing your own meat





Terry S. Singeltary Sr.

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