Estimating relative CWD susceptibility and disease progression in farmed white-tailed deer with rare PRNP alleles.

Chronic wasting disease is a prion disease affecting both free-ranging and farmed cervids in North America and Scandinavia. A range of cervid species have been found to be susceptible, each with variations in the gene for the normal prion protein, PRNP, reportedly influencing both disease susceptibility and progression in the respective hosts. Despite the finding of several different PRNP alleles in white-tailed deer, the majority of past research has focused on two of the more common alleles identified-the 96G and 96S alleles. In the present study, we evaluate both infection status and disease stage in nearly 2100 farmed deer depopulated in the United States and Canada, including 714 CWD-positive deer and correlate our findings with PRNP genotype, including the more rare 95H, 116G, and 226K alleles. We found significant differences in either likelihood of being found infected or disease stage (and in many cases both) at the time of depopulation in all genotypes present, relative to the most common 96GG genotype. Despite high prevalence in many of the herds examined, infection was not found in several of the reported genotypes. These findings suggest that additional research is necessary to more properly define the role that these genotypes may play in managing CWD in both farmed and free-ranging white-tailed deer, with consideration for factors including relative fitness levels, incubation periods, and the kinetics of shedding in animals with these rare genotypes.

Introduction

Chronic wasting disease (CWD) is a progressive neurologic disease of cervids caused by a transmissible, misfolded protein—the prion protein. [1,2] The disease is naturally occurring in white-tailed deer (Odocoileus virginianus), mule deer (Odocoileus hemionus), Rocky Mountain elk and red deer (Cervus elaphus sspp.), moose (Alces alces), and reindeer (Rangifer tarandus). [3] It has been reported in farmed and free-ranging cervids in 26 US states, 3 Canadian provinces, the Republic of Korea, Norway, Sweden, and Finland. [3-8] Chronic wasting disease is highly transmissible through direct contact or environmental contamination, and has been detected at various levels in all tissues and bodily fluids of cervids examined to date. [9-18]

The misfolded protein—commonly designated “PrPres” due to its resistance to harsh physical treatments, is derived from the cellular prion protein—“PrPC”—a normal protein encoded by the PRNP gene, which is present in a range of animals in the phylum Chordata. [19-21] Prion disease transmission and pathogenesis relies on the coerced conversion of normal PrPC by PrPres into the abnormally folded isoform, which collects in the form of amyloid in a variety of tissues, most notably the central nervous system, resulting in the eventual demise of the host. The tertiary structure of this misfolded protein, and its properly folded counterpart, is inherently dependent on its primary amino acid sequence. [22-30] As such, the ability of the misfolded protein to coerce normally folded prion proteins into an abnormal, amyloid-forming structure is highly dependent on the primary amino acid sequence of both the infectious and host prion proteins. Significant variation between host and infectious prion proteins results in reduced host susceptibility, and in some cases complete resistance to disease—a phenomenon known as the “species barrier” when considering natural or experimental inter-species transmission of the infectious prion agent. [31-35]

Variations in prion disease susceptibility have been reported across most species naturally affected by these agents. Humans with variation in amino acids at either position 127 or 129 are resistant to various transmissible forms of Creutzfeldt-Jakob disease and Kuru. [36] Goats with amino acid variations present at positions 146, 211, and 222, as well as several other sites, show reduced susceptibility to either BSE or sheep scrapie. [24,37,38] Sheep with variations at position 136, 154, and 171, among others, present with a range of susceptibilities to classical scrapie—including, in the case of A136R154R171 homozygous sheep, near-complete resistance to infection. [39-41] The latter finding has led to a multinational effort to breed sheep towards resistance to classical scrapie infection in areas where the disease is endemic, resulting in a significant decline and near-eradication of the disease in countries employing targeted breeding programs. [42-44]

Polymorphisms in the PRNP gene of white-tailed deer, mule deer, elk, fallow deer and reindeer have all been found to influence susceptibility to CWD in wild, farmed, and experimental populations. [26,45-49] The low prevalence of CWD in these populations has often made it difficult to adequately understand the role these polymorphisms may play in the disease process. Additionally, many of these studies only incorporate a binary (positive or not detected) approach to disease diagnosis, and fail to include disease staging as a factor in susceptibility. [45,50-53] Lastly, and perhaps most importantly, most of these polymorphisms are quite rare, and animals homozygous for these alleles, or in rare heterozygous combinations, have neither been observed in CWD endemic populations nor tested for their susceptibility following natural exposure. [26] Exceptions include the 225F polymorphism in mule deer and the 132L polymorphism in Rocky Mountain elk. In the case of 225FF homozygous mule deer, a small group of animals placed on a heavily contaminated pasture eventually developed progressive neurologic disease and neuropathology characteristic of CWD, although the 225F allele seems to be a significant barrier to infection in wild populations under more typical exposure conditions. [45,54] Elk heterozygous or homozygous for the 132L polymorphism likewise show reduced susceptibility in both wild and captive populations, however 132LL homozygous elk have only rarely been found to be infected. [48,55]

In the present study, we sought to better define the relative susceptibilities of white-tailed deer heterozygous and homozygous for several different PRNP alleles, including 95H, 96G and 96S, 116G, and 226K. Samples were analyzed from nearly 2100 farmed deer depopulated following exposure to CWD, including 714 deer infected with CWD, with postmortem prevalence ranging from 6–83% across 20 separate herds in the United States and Canada. In addition to CWD status, we also examined the correlation of PRNP genotype with the stage of disease, ranging from one (detection in retropharyngeal lymph nodes, RLN, only) to five (detection in RLN in addition to significant immunostaining in the obex region of the brainstem). Finally, we surveyed 117 healthy white-tailed deer herds in the United States and 7 white-tailed deer herds in Canada to assess the distribution of these five different alleles across North American farmed deer populations. We hypothesized that CWD status and disease stage would be most significant and severe in animals homozygous for the 96G allele, with other pairings less significantly and severely affected. We also hypothesized that allele frequencies would vary between the Canada and the United States, and within geographic regions of the United States. We found several combinations of alleles that were associated with significantly reduced CWD prevalence and/or disease severity, and that specific alleles may be more common in different regions of North America. These findings suggest that variations in susceptibility to CWD may play a role in managing the disease in this species, and those variations may be more common in farmed deer in certain areas, warranting further exploration of PRNP markers in the natural white-tailed deer host.

Methods

Ethics statement

This study was conducted retrospectively, using tissues collected from animals depopulated during the normal course of disease control efforts by the United States Department of Agriculture and the Canadian Food Inspection Agency. Sequencing of PRNP genotypes in healthy animals was conducted on a contract testing basis, with results anonymized and with the consent of the owners submitting the samples.

Study population

Twenty white-tailed deer herds depopulated in the United States (11 herds with 1185 adult animals) and Canada (9 herds with 906 adult animals) were included in the analysis. Each herd had initially reported one or more deer with a positive diagnosis of CWD, and was subsequently placed under quarantine. When the animals were later depopulated, a variety of samples were collected, including RLN and the obex region of the brainstem for conventional CWD testing, and either blood or ear punch for PCR amplification and sequencing of the PRNP gene. All herds were depopulated in roughly the past 5–10 years, though not all herds depopulated in those years had samples available, and in some cases samples were not available from all animals in their respective herds. Missing samples included animals too young to test, animals from which a sample was otherwise unavailable, and animals with poor quality DNA samples. Animal age was generally unknown, though only adult animals over 1 year of age were considered for the study. Of the 2091 animals evaluated, 714 were ultimately found to be CWD positive (34.1%). Further details on the herd sizes, their country of origin, gene frequencies, and CWD prevalence (based on results from cases with available DNA) can be found in Tables 1 and 2.

Table 1

Summary of herds in the United States providing samples for the present study.

Eleven herds in the United States, comprised of 1185 samples from individual deer were included in the analysis. Prevalence and genotype data from each herd, based on animals for which both genetic data and CWD status are available, are shown.

Herd ID	Number present	Number available for testing	CWD Prevalence (%)	Genotype
				96GG		96GS		96SS		95H/96G		95H/96S		95HH		96G/226K		96S/226K
				-	+	-	+	-	+	-	+	-	+	-	+	-	+	-	+
A	81	80	12.5	36	7	22	3	5	0	0	0	0	0	0	0	6	0	1	0
B	99	96	9.4	30	4	38	3	12	1	1	0	0	0	0	0	5	1	1	0
C	47	47	12.8	19	4	15	2	7	0	0	0	0	0	0	0	0	0	0	0
D	140	140	5.7	68	8	41	0	12	0	5	0	3	0	0	0	2	0	1	0
E	129	128	19.5	44	23	33	1	6	0	11	0	0	0	2	0	5	1	2	0
F	99	99	9.1	60	3	25	6	3	0	0	0	0	0	0	0	2	0	0	0
G	85	79	26.6	51	20	7	1	0	0	0	0	0	0	0	0	0	0	0	0
H	116	116	22.4	25	20	41	5	21	0	0	0	0	0	0	0	2	1	1	0
I	18	14	35.7	2	5	5	0	0	0	0	0	1	0	0	0	1	0	0	0
J	356	356	79.8	7	120	37	138	24	19	3	5	0	0	0	0	0	2	1	0
K	36	30	20.0	16	4	5	2	1	0	1	0	0	0	0	0	0	0	1	0
Total	1206	1185	34.5	358	218	269	161	91	20	21	5	4	0	2	0	23	5	8	0

Table 2

Summary of herds in Canada providing samples for the present study.

Nine herds from Canada, comprised of 906 samples from individual deer, were included in the analysis. Prevalence and genotype data from each herd, based on animals for which both genetic data and CWD status are available, are shown.

Herd ID	Number present	Number available for testing	CWD Prevalence (%)	Genotype
				96GG		96GS		96SS		95H/96G		96G/116G		96S/116G		116GG
				-	+	-	+	-	+	-	+	-	+	-	+	-	+
L	72	43	30.2	15	11	7	2	0	0	0	0	6	0	0	0	2	0
M	29	29	82.8	0	11	4	12	1	1	0	0	0	0	0	0	0	0
N	56	55	23.6	16	8	18	1	3	0	0	0	4	4	1	0	0	0
O	179	133	8.3	67	8	32	3	9	0	0	0	11	0	3	0	0	0
P	325	241	58.1	28	82	45	36	6	2	0	1	13	17	8	2	1	0
Q	23	12	41.7	3	3	2	1	1	0	0	0	1	1	0	0	0	0
R	70	47	63.9	0	9	10	19	6	2	0	0	1	0	0	0	0	0
S	66	35	11.4	10	1	12	1	1	0	0	1	7	1	0	0	1	0
T	414	311	20.9	110	47	6	1	0	0	0	0	106	17	8	0	16	0
Total	1264	906	33.8	249	180	136	76	27	5	0	2	149	40	20	2	20	0

Unaffected populations

Samples from healthy animals in 117 herds in the United States (n = 6030 animals) and 7 herds in Canada (n = 1313 animals) were also evaluated for PRNP genotype frequencies. Herds in the United States were further subcategorized by region, and included 75 herds from the Midwest (n = 3865 animals tested from Iowa, Indiana, Michigan, Minnesota, Missouri, North Dakota, Ohio and Wisconsin), 29 from the Northeast (n = 1651 from Pennsylvania), and 13 from the South (n = 514 from Texas and Alabama). Although it was common for entire herds to be included in the analysis, it is important to note that herds submitting samples for testing were not likely to be random and the number of states, and herds included, in each region varied. A summary of allele frequencies in white-tailed deer herds in the United States and Canada may be found in Table 3. Genotypic data are also provided in S1 Table.

Table 3

Summary of genotype frequencies in healthy North American white-tailed deer herds.

Data from whole herds opting to perform PRNP genotyping were included in the analysis, which found significant differences in distribution between Canada and the United States, as well as between specific regions of the United States.

Location	Number of Herds	Number of Animals	Allele Frequency %
			95H	96G	96S	116G	226K
United States
Midwest	75	3865	1.5	72.6	22.1	0	3.6
Northeast	29	1651	3.1	71.5	21.1	0	4.1
South	13	514	0	58.1	39.2	0	2.7
United States Total	117	6030	1.8	71.0	23.3	0	3.7
Canada
Alberta	4	629	0.56	67.1	29.8	2.5	0
Saskatchewan	2	684	2.2	62.9	31.4	3.3	0
Canada Total	6	1313	1.4	65.0	30.7	2.9	0

PRNP analysis

For CWD correlation, nucleic acids were extracted in most cases from whole blood samples preserved in EDTA, or in some cases ear punch biopsies, using a conventional DNA extraction kit. (ThermoFisher, USA) For healthy herd gene frequencies, DNA was most commonly extracted from hair samples provided by healthy herds across North America, though semen, antler core, ear notches and other biopsy samples were also included. Data from these healthy herds solely included locations where the entire herd was sampled. An approximately 750bp PRNP gene sequence was amplified by conventional PCR and sequenced as previously described. [46,56] PCR sequences were aligned and evaluated using Geneious software version 10.2 (www.Geneious.com). Specific single nucleotide polymorphisms at position 95 (glutamine [Q] or histidine [H]), 96 (glycine [G] or serine [S]), 116 (alanine [A] or glycine), and 226 (glutamine or lysine [K]) were identified and recorded.

Immunohistochemistry of retropharyngeal lymph node and brainstem

Retropharyngeal lymph node and brainstem tissues were examined microscopically for PrPCWD immunostaining as previously described. [56,57] Briefly, tissue was preserved in 10% neutral buffered formalin and subsequently embedded in paraffin blocks. Tissue sections 5 μm thick were mounted on glass slides and deparaffinized before treatment with 95% formic acid. Immunohistochemical staining for PrPCWD was performed with the primary antibody anti-prion 99 (Ventana Medical Systems, Tucson, AZ) and then counterstained with hematoxylin. The obex sections were scored from 0 to 4 on the basis of the following criteria: grade 0, no IHC staining observed within the obex; grade 1, IHC staining only within the dorsal motor nucleus of the vagus (DMNV); grade 2, IHC staining within the DMNV and area postrema with or without focal staining in the nucleus of the solitary tract (NST) and adjacent white matter; grade 3, IHC staining in the DMNV and NST with light to moderate staining extending into other nuclei and white matter; grade 4, heavy IHC staining of the DMNV, multiple other nuclei, and white matter throughout the obex. Results were tabulated according to RLN and obex immunostaining, with individuals exhibiting immunostaining in the RLN alone scored as a “1,” while those with additional immunostaining in the obex scored as 2–5 depending on obex staining intensity. As with previous studies, all deer that had obex staining always concurrently had staining in the RLN, a finding characteristic of CWD in white-tailed deer.

Statistical analyses

Statistical analysis was done using R version 3.5.1 with the brms [58] and nlme [59] packages. A linear mixed model, with herd included as a random effect, was used to calculate coefficients for disease stages relative to the 96GG genotype with associated 95% confidence intervals. A Bayesian mixed effects logistic regression model with herd again included as a random effect was used to determine odds ratios of infection in various genotypes relative to the 96GG genotype. A weakly informative prior for genotypes was defined as the Cauchy distribution with location and scale parameters of 0 and 2.5, respectively. This approach accounted for differences in disease prevalence and genotypic distribution between and across farms in an effort to better estimate relative susceptibility and disease progression. The Markov-chain Monte-Carlo (MCMC) sampling was used with 500000 iterations, following an initial burn-in period of 5000 iterations. The scale reduction factor was calculated to assess convergence and adequate mixing of the chains. The posterior medians and 95% credible intervals were used for inference.

In order to predict outcomes for genotypes that were not observed, an additive mixed effects model, both linear and logistic, were built using data from measured allele pairs to estimate the contribution of each single allele. The prediction interval for the log odds estimate was calculated using the merTools package [60] and is done by drawing a sampling distribution for the random and fixed effects and then estimating the fitted value across that distribution. The calculated interval includes all variation in the model except for variation in the covariance parameters.

A chi-squared test was used to compare PRNP frequencies between Canada and the United States, as well as between different regions of the United States.

Results

Correlation of PRNP genotype with CWD infection status

Positive and negative CWD infection status were correlated to PRNP genotypes using the 96GG genotype as a reference point to assess odds ratios of infection. A significant reduction in odds ratio of infection was seen with all genotypes examined, except for the 96G/226K genotype. While there was a trend towards reduced odds ratios in this genotype, the findings were not statistically significant. Among animals heterozygous for the 96G allele, odds ratios were lowest in animals carrying the 95H allele (0.257, 95% CI: 0.08–0.80), though the results were not significantly different than those found in animals with the 96GS genotype (0.319, 95% CI: 0.23–0.43). Among alleles for which sufficient data were available for modeling, animals homozygous for the 116G allele had the lowest odds ratio of being found infected (3 x 10−6), though confidence intervals ranged widely. Results are summarized in Table 4 and Fig 1. Modeling odds ratios of infection in non-96G homozygous genotypes continued to exhibit wide-ranging confidence intervals, though suggested that 95HH homozygous genotypes in particular may have the lowest odds ratios for being found CWD positive (Fig 2).

Table 4

Relative CWD susceptibility and disease staging in white-tailed deer with rare alleles, in reference to the 96GG genotype.

Odds ratio of identifying infection in rare alleles was determined using Bayesian mixed effects logistic regression, while relative disease stages were calculated using linear coefficient modeling. Significantly lower odds of being found infected, relative to the 96GG genotype, were observed in all rare genotypes except for the 96G/226K genotype, where findings were suggestive of lower odds ratios, though statistically inconclusive. Negative values for disease staging indicate a trend towards earlier stages of disease, and a significantly lower disease stage was found in all rare genotypes evaluated relative to animals with the 96GG genotype.

Genotype	Bayes Logistic OR	Logistic 95% CI	Linear Coefficient	Linear 95% CI
96GS	0.319	(0.23, 0.43)	-0.839	(-0.96, -0.72)
96SS	0.069	(0.04, 0.12)	-1.502	(-1.72, -1.29)
95H/96G	0.257	(0.08, 0.80)	-1.205	(-1.66, -0.75)
96G/116G	0.440	(0.28, 0.68)	-0.463	(-0.67, -0.26)
96G/226K	0.551	(0.18, 1.39)	-0.828	(-1.28, -0.38)
96S/116G	0.090	(0.02, 0.36)	-1.130	(-1.63, -0.63)
116GG	0.000003	(0.00, 0.30)	-0.853	(-1.39, -0.32)
96S/226K	0.00005	(0.00, 0.68)	-1.137	(-1.96, -0.31)
95H/96S	0.018	(0.00, 2.56)	-0.744	(-1.92, 0.43)

Fig 1

Summary of log odds ratios of white-tailed deer with 96G heterozygous and 96SS homozygous genotypes being found CWD positive, and the stage of disease recorded among those infected relative to the 96GG genotype.

The most common genotypes found in the study are presented, showing that all heterozygous 96G crosses exhibit some level of slowed disease progression and/or reduced susceptibility.

Fig 2

Estimates of log odds ratios and disease staging for the 96S, 116G, 226K, and 95H alleles in the homozygous state.

Using data from measured allele pairs, an additive mixed effects model was developed to predict outcomes in genotypes with insufficient data. Predicted estimates for disease susceptibility and progression are show for both heterozygous 96G genotypes and homozygous pairings.

Correlation of PRNP genotype with CWD infection stage

Disease stages were correlated to rare PRNP genotypes, again using the 96GG genotype as a reference point to evaluate differences in disease severity. In all genotypes examined, a significant reduction in disease staging was observed compared to the 96GG reference genotype. As noted with odds ratios above, the most significant reduction in disease staging was observed in animals with the 95H/96G genotype (-1.205, 95% CI: -1.66 to -0.75), though again this finding was not significantly different than what was observed for 96GS heterozygous animals (-0.839, 95% CI: -0.96 to -0.72). Among homozygous animals with sufficient data available for modeling, disease staging was lowest in animals with the 96SS genotype, though it should be noted that low or absent numbers of rarer genotypes made their analysis challenging. Results again are summarized in Table 4 and Fig 1, with models addressing disease progression in other homozygous genotypes again presented in Fig 2.

Frequency of PRNP alleles in healthy farmed white-tailed deer herds

Significant differences were observed in the frequency of various alleles in Canadian and US herds—particularly with regard to the exclusive presence of the 116G allele in Canadian herds and the 226K allele in US herds. The 96G allele was found to be at a significantly higher frequency in US herds, while the 96S allele was found to be at significantly higher frequencies in Canadian herds. Within the United States, significant differences in PRNP frequencies were also observed between different regions of the country, regions that are admittedly arbitrary with samples available only from some states within those regions. Most notably, the 95H allele was significantly more common in herds in the Northeast compared to both Midwestern and Southern herds, while the 96S allele was found at a higher frequency in Southern states compared to herds in the Midwest and Northeast. No differences in allele frequencies were observed between herds in the Canadian provinces of Alberta and Saskatchewan. (Table 3 and S1 Table)

Because the samples from healthy deer herds in both the United States and Canada are presumed to have been submitted non-randomly—e.g. those herds financially capable of testing, and those having a particular interest in PRNP genotyping, it is important to note that these findings should be interpreted with caution.

Discussion

A significant amount of research over the past two decades has been conducted on PRNP gene frequencies in both wild and farmed white-tailed deer populations affected by CWD, which cumulatively has led to the understanding that animals with different PRNP alleles are differentially susceptible to CWD infection. [8,26,50,51,57] Recent research has pointed to slower disease progression in animals with several of the more common genotypes, notably those carrying the 96S allele, in addition to their reduced susceptibility. [56,57] Each of these previous studies, however, have suffered from limitations which may hinder broader interpretation, including low disease prevalence and/or negligible or absent populations of animals representing rarer genotypes. [50-52,61] The present study represents one of the largest in-depth evaluations of the relationship between PRNP genotype and both CWD status and disease stage in white-tailed deer, and the relatively high disease prevalence in many of these populations provided us with important insight into susceptibility and disease progression in some of the more rare genotypes.

Previous studies have typically focused on two of the most common alleles—commonly referred to as the 96G and 96S alleles, and the corresponding 96GG, 96GS, and 96SS genotypes. Occasionally these studies make use of genotyping strategies that might ignore the contribution of other, rarer alleles. [57,62] In the present study, as in past studies, the 96G and 96S alleles made up a substantial percentage of total alleles in a population, making statistical comparisons easier even with small population sizes. [26,56] We found that, in line with previous studies, animals with the 96GS and 96SS genotypes were at a significantly reduced risk of being found CWD positive at the time of depopulation, and were generally in a significantly earlier stage of disease when infected compared to animals with the 96GG genotype.

We extended our analyses to rarer alleles, including the 95H, 116G, and 226K alleles, which to date have only garnered passing interest in susceptibility studies. [46,50,61,63] We report that the animals evaluated in this study with the 95H/96G and 96G/116G genotypes not only appear to face significantly lower risk of being found CWD positive, they, like their 96GS and 96SS counterparts, were also found to be in significantly earlier stages of disease at the time of depopulation. While there was a trend towards reduced susceptibility in animals with the 96G/226K genotype, their differences compared to animals with the 96GG genotype were not statistically significant. The 96G/226K genotype was, however, found to correlate with significantly lower disease scores than 96GG homozygous animals in the study. Models extending available data to 95HH, 116GG, and 226KK homozygous genotypes suggest the potential for an even further reduction in both susceptibility and disease progression.

To a limited extent, both the 95H and 116G alleles have been evaluated in prior studies for CWD susceptibility in either free-ranging or farmed white-tailed deer herds. A study of a wild deer population in Illinois found that animals with the 95H allele faced a risk of being found CWD-positive 1/5th that of the herd at large, similar to data reported in the present study (OR = 0.257, Table 4). [51] A limited bioassay study including two animals with the 95H allele found that CWD incubation periods were nearly double that of their 96GG and 96GS counterparts. [61] Subsequent examinations of animals in that report suggested differences in CWD prion protease sensitivity which might affect diagnostic test results—an important factor to consider when evaluating the results from the present study. [64] An evaluation of a farmed herd in Nebraska, meanwhile, found that white-tailed deer with the 116G allele were roughly half as likely to be found CWD positive compared to the herd at large, again very similar to the results reported here (OR = 0.440, Table 4). [46] Little information is available regarding the 226K allele in the natural host; however, in vitro misfolding studies have shown that, like several other rare cervid PRNP alleles, recombinant 226K prion protein is significantly limited in its ability to misfold in the presence of CWD prions. [65] Additional work is needed to more adequately define relative infection odds ratio and disease staging in not only the 96G/226K genotype, but other rare alleles as well—especially in animals homozygous for 95H, 116G, or 226K alleles.

While our findings, and those of past research efforts, suggest that deer with specific alleles face a significantly lower risk of being found CWD positive at depopulation—as well as a significant deceleration in disease progression when infected—it seems likely that deer carrying these alleles are not completely resistant to the disease. It is therefore uncertain what role, if any, PRNP genetics may play in the management of CWD in both farmed and free-ranging deer. From a diagnostic perspective, animals with more susceptible alleles exhibit a more rapid progression of the disease, and are thus more readily identified on antemortem testing. This particular factor may prove helpful in more quickly identifying infected herds and placing them under quarantine. [56,57] The increased diagnostic sensitivity offered by animals with susceptible genotypes, however, should be carefully weighed against the drawbacks of raising highly susceptible animals, especially in areas where CWD is highly endemic.

Apart from the diagnostic challenges noted above, additional factors that should be considered include the role that less susceptible alleles may have on general animal health, any delays in disease progression, and the resultant kinetics of prion shedding in infected animals carrying them. At present, there is almost no objective information available on the fitness of various PRNP genotypes in cervids [54], and while there are several limited reports of CWD prion shedding in more common white-tailed deer genotypes [66-68], the biological relevance of prions likely shed in biological fluids has proven more difficult to assess. [10,15,69] The lifespan of the host is also relevant when considering incubation periods of the disease—particularly in farmed deer, where age may be useful as a selective management factor, similar to strategies used to address concerns for zoonotic transmission of BSE from cattle. [70] Lastly, it is critical to understand the mutable nature of the CWD prion agent itself in the face of shifting host genetic background, and whether any novel strains that may arise have any notable differences in disease manifestation and zoonotic potential. [71-74]

In free-ranging herds, it is even less clear if there is a role for human intervention, and more importantly whether CWD may be actively shaping PRNP allele frequencies in wild populations. [26] At least one study has found that the less susceptible 96S allele may provide a significant fitness advantage in a CWD endemic area, making it especially valuable to reevaluate the current frequencies of PRNP alleles in areas hard hit by the CWD epidemic. [62] As with farmed deer, understanding the relationship between PRNP genotype, fitness, prion shedding, and incubation periods would prove useful to those seeking to manage the disease in wild herds as allele frequencies shift over time. Our surveillance efforts in farmed populations shows that rare alleles are fairly well distributed across North America, with potential regional variation in frequencies, and similar efforts in wild cervids in both North America and Scandinavia may prove both useful and informative.

In summary, we provide further evidence that specific and often rare PRNP alleles of white-tailed deer appear to correlate strongly to both CWD susceptibility and progression. Though rare, these alleles may be found in farmed deer herds across the United States and Canada, with potential, as yet unexplained, regional variations observed. Ongoing studies in farmed deer should provide some insight into both the relative fitness of animals carrying these alleles and their utility in managing CWD in endemic areas. The role these genotypes may have in managing the disease in free-ranging white-tailed deer should likewise continue to be explored, within the context of those considerations noted above.

Alternative presentation of Table 2, to present genotype data from healthy US and Canadian white-tailed deer herds.

(DOCX)

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30 Oct 2019

PONE-D-19-28154

Estimating relative CWD susceptibility and disease progression in farmed whitetail deer with rare PRNP alleles

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Academic Editor

PLOS ONE

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Reviewer #2: Yes

Reviewer #3: Partly

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Reviewer #2: Yes

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Reviewer #1: Haley et. al provide data regarding PRNP genotype influence on CWD susceptibility and disease progression in whitetail deer from captive facilities. Over 2000 animals were tested in the study, finding more than 700 CWD+ cases from numerous captive deer facilities. Importantly, many rare genotypes are represented in this study. The amount of time and effort to complete such a study is commendable. Such a large test cohort was critical in making more meaningful statistical comparisons with rare alleles. Clearly showing that there are several polymorphisms that can confer higher resistance to CWD is very encouraging. I have a few minor comments that I will list below.

Lines 187-189. I think a reference to Figure 2 should be included here as that pertains to the modelling described.

Line 197. Insertion of "homozygous" between among and animals would help keep the reader clear on which genotype status is being discussed.

Table 3. In addition to the allelic frequencies provided in tables 1&2, I think it would be nice to include a column in table 3 with the actual number of deer having each listed genotype. The text often alludes to how rare some of them were, including this number would prevent the reader from having to do the math themselves (allelic frequency x the number of deer sampled).

Figure 2, upper right. Should 116GG also have a red dot for the observed outcome? Table 3 and the text in line 185 imply that such an animal was included in the study. Maybe IHC was not available for this/these deer?

Figure 2 legend. Typo. Final sentence "show" should be shown.

Reviewer #2: This paper summarizes genetic and diagnostic data from a sizable number of captive white-tailed deer herds to describe genetic influences on prion disease occurrence and progression within infected animals. As noted, the findings generally reaffirm observations and patterns reported in cited references as well as in other publications not cited here. The approach appears to be technically sound and the paper well-organized & -written.

Two corrections do need to be made:

1. The common name for this species is white-tailed deer (NOT "whitetail"). This is consistently wrong throughout the draft text, tables, and figures.

2. The term "data" is plural (for datum), and associated verbs should be corrected accordingly throughout.

Reviewer #3: This manuscript compares the Prnp genotypes of white-tailed deer with infection status to determine whether there are genotype affects susceptibility/resistance to CWD infection. The genotypes and CWD status are from farms depopulated by both the USDA and the CFIA. The authors make a number of conclusions based on this data---particularly that no genotype appears to be resistant to CWD infection, some genotypes, however, result in longer disease progressions. Uninfected herds were also analyzed to determine the genotypic variability. Of interest, 226K was found only in US white-tailed deer while 116G was found only in Canadian deer. The authors also report regional differences in allele frequencies in the captive white-tailed deer farms.

1. For the susceptibiity/resistance to CWD; it would be helpful to link CWD status in a given farm to genotype---or at least associate prevalence of CWD to CWD status for the different genotypes. It would seem likely that infection rates may overcome or, at least, impact resistance to disease---on the other hand, farms with very low infection rates may not have resulted in enough animals being infected to ensure a heterozygotes or deer that are homozygous for alleles other than 96G have been infected.

2. The authors state in the abstract that certain genotypes were not infected in some high prevalence farms. I could not find this data. This is critical information with respect to determining whether genetics play a role in susceptiblity.

3. For the regional variation in the uninfected herds---this is very interesting as the movement of cervids over the decades would likely make captive herds more similar. Are all of these samples from herds that no longer import/move deer? Was an entire herd samples, or just a subset of the animals (which could lead to a bias in the data analysis).

4. The 116G/226K data is again very intriguing---for the same reasons as mentioned above. Is there significant geographic distance between the herds analyzed in the US and those in Canada? At least with free-ranging animals, there tend not to be significant differences in allele frequencies over long geographic ranges.

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Reviewer #2: No

Reviewer #3: No

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13 Nov 2019

Public Library of Science ONE

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Dear Dr. Caughey,

We would like to take the opportunity to thank you for your time, and that of the reviewers, in providing feedback on our manuscript entitled “Estimating relative CWD susceptibility and disease progression in farmed whitetail deer with rare PRNP alleles.” The feedback has proven very helpful in improving the manuscript, and our revisions to each comment are provided below. We first provide revisions to the Funding and Competing Interests Statements as requested:

Funding Statement:

Simpson Whitetails Genetic Testing provided support in the form of salaries for authors (DS and AC), as well as genotypic data from a subset of CWD negative herds, representing approximately 50% of the healthy herds reported. These authors had no role in study design or data analysis. The specific roles of these authors are further articulated in the “author contributions” section.

Competing Interests Statement:

Two authors (DS and AC) were employed by Simpsons Whitetails Genetic Testing at the time this study was undertaken. These two authors provide a commercial test for PRNP genotyping to the deer and elk farming industry. There are no patents or products in development associated with this commercial venture. This association does not alter our adherence to PLOS ONE policies on sharing data and materials.

Response to reviewers:

Reviewer #1

1. The reviewer suggests that a reference to Figure 2 be included with the statistical modelling methodology.

Response: We have added the reference as suggested.

2. The reviewer suggests that the qualifier “homozygous” be inserted to help clarify with genotypes are being discussed.

Response: We have added the qualifier “homozygous” as suggested.

3. The reviewer suggests a revision to the Tables to include information on the number of deer with each listed genotype.

Response: For this response, we chose to focus on Table 1, to include information on genotype and CWD status. We felt that this best addressed comments by reviewer 1 and reviewer 3. We have also included a supplemental Table S1 which presents genotype data for healthy herds (vs. allele frequency for Table 2).

4. The reviewer asks whether 116GG should have an observed outcome in Figure 2; as they astutely observed, the data was present in the manuscript.

Response: The modeling data for 116GG animals is available as the reviewer notes, as shown in Table 3. The relatively low number of these animals in the study led to a fairly wide error range, outside of the scale of the graphs which were practical for the Figure (-6 log vs. -4 log range for the graphs). Because of this, the observed outcome data point for these animals (which did have IHC data available) was left out of the figure for the sake of clarity. We have provided clarification in the figure legend to point out that the data is available in Table 3. As additional data is collected on these and other animals in the future, we may be able to provide further information on how well our model fit for these graphs for homozygous animals.

5. The reviewer identified a grammatical error in the legend for Figure 2.

Response: We have corrected this error in the final manuscript.

Reviewer #2

1. The reviewer notes that the common name for the species reported is “white-tailed deer.”

Response: We have amended all occurrences of “whitetail deer” to “white-tailed deer” throughout the manuscript.

2. The reviewer points out that data is a pleural noun, and that verbs throughout the manuscript should be corrected appropriately.

Response: We have corrected the errors throughout the manuscript.

Reviewer #3

1. The reviewer suggests that our analysis associates prevalence of CWD to CWD status for different genotypes, to address the influence of prevalence on disease resistance.

Response: The authors agree, and in fact that information is built into our modeling approach. Since this was not clear in our description of the methods, we have included clarification in our revised manuscript as follows:

“This approach accounted for differences in disease prevalence and genotypic distribution between and across farms in an effort to better estimate relative susceptibility and disease progression.”

2. The reviewer notes that we report that some genotypes were not infected on some of the higher prevalence farms, and notes the absence of data presented which specifically illustrates this.

Response: We agree that the tables we’ve provided do not allow the reader to fully appreciate the frequency of various genotypes present – as Reviewer 1 also suggested. In response to this, we’ve modified Table 1 to include genotype data instead of allele frequency and CWD status, and we have included a supplemental Table 1 that covers genotypic data for healthy herds.

3. The reviewer asks whether the healthy herds that were evaluated are currently importing or moving deer, and whether an entire herd was sampled for the analysis.

Response: Many of the healthy herds included in our analysis are importing and exporting deer, semen, and in some cases embryos. A small subset of these herds are entirely closed and self-sufficient. The analysis solely includes data from herds where all animals were available for genotyping, to avoid bias as the reviewer notes. We have made note of this in the methods section as follows: “Data from these healthy herds solely included locations where the entire herd was sampled”

4. The reviewer finally asks whether there was a significant distance between the Canadian and United States herds evaluated, which might offer some insight into the distribution of 116G and 226K alleles.

Response: The geographic distance between the nearest Canadian and US herds is roughly 400 miles, however the question likely goes beyond geographical distance and more likely lies in sire selection/preferences and international laws covering the movement of deer and deer products (e.g. semen). It is worth noting that historically, the 116G allele has been reported on a farmed deer herd depopulated in Nebraska (O’Rourke et al, JGV, 2004), and this herd was rumored to be owned by a Canadian deer farmer who may have moved deer between the US and Canada prior to regulations preventing international movement of cervids. We feel that it is likely that either of these currently unreported alleles may eventually be found in their respective US or Canadian herds, however it is also likely their frequencies will be quite low.

We submit the revised manuscript, included a copy with changes highlighted, for your further consideration for publication in PLOS One, and sincerely thank you for your assistance and contributions.

Sincerely yours,

Nicholas James Haley

Kahla Merrett

Amy Buros-Stein

Dennis Simpson

Andrew Carlton

Antanas Staskevicius

Tracy Nichols

Aaron Lehmkuhl

Bruce Thomsen

Submitted filename: PLOS One Response to criticisms.docx

Click here for additional data file.

15 Nov 2019

Estimating relative CWD susceptibility and disease progression in farmed whitetail deer with rare PRNP alleles

PONE-D-19-28154R1

Dear Dr. Haley,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Byron Caughey

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

20 Nov 2019

PONE-D-19-28154R1

Estimating relative CWD susceptibility and disease progression in farmed white-tailed deer with rare PRNP alleles

Dear Dr. Haley:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Byron Caughey

Academic Editor

PLOS ONE