https://cpw.cvlcollections.org/files/original/e84c6ef9c96c721dad6eb8f497c34774.pdf 6e14d5a1cc99ffd5a3492c6df8893f4a PDF Text Text The research in this publication was partially or fully funded by Colorado Parks and Wildlife. Dan Prenzlow, Director, Colorado Parks and Wildlife • Parks and Wildlife Commission: Marvin McDaniel, Chair • Carrie Besnette Hauser, Vice-Chair Marie Haskett, Secretary • Taishya Adams • Betsy Blecha • Charles Garcia • Dallas May • Duke Phillips, IV • Luke B. Schafer • James Jay Tutchton • Eden Vardy �viruses Article Feline Foamy Virus is Highly Prevalent in Free-Ranging Puma concolor from Colorado, Florida and Southern California Sarah R. Kechejian 1 , Nick Dannemiller 1 , Simona Kraberger 2 , Carmen Ledesma-Feliciano 3 , Jennifer Malmberg 4 , Melody Roelke Parker 5 , Mark Cunningham 6 , Roy McBride 7 , Seth P. D. Riley 8 , Winston T. Vickers 9 , Ken Logan 10 , Mat Alldredge 11 , Kevin Crooks 12 , Martin Löchelt 13 , Scott Carver 14 and Sue VandeWoude 1, * 1 2 3 4 5 6 7 8 9 10 11 12 13 14 * Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; skecheji@colostate.edu (S.R.K.); dannemillern@gmail.com (N.D.) Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA; simona.kraberger@gmail.com Division of Infectious Diseases, University of Colorado Anschutz Medical Campus, 12700 E 19th Ave, Aurora, CO 80045, USA; Carmen.Ledesma_Feliciano@colostate.edu Wyoming State Vet Lab, University of Wyoming, 1174 Snowy Range Road, Laramie, WY 82072, USA; jennifer.malmberg@uwyo.edu Frederick National Laboratory of Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21701, USA; melody.roelke-parker@nih.gov Florida Fish and Wildlife Conservation Commission, 1105 SW Williston Road, Gainesville, FL 32601, USA; mark.cunningham@myfwc.com Rancher’s Supply Inc., Alpine, TX 79830, USA; livestockprotection@gmail.com National Park Service, Santa Monica Mountains National Recreation Area, Thousand Oaks, CA 90265, USA; seth_riley@nps.gov Karen C. Drayer Wildlife Health Center, University of California, Davis, CA 95616, USA; twvickers@ucdavis.edu Wildlife Researcher Colorado Parks and Wildlife, 2300 S. Townsend Avenue, Montrose, CO 80203, USA; ken.logan@state.co.us Colorado Division of Wildlife Office, Mammals Research, 317 W. Prospect Rd, For Collins, CO 80526, USA; Mat.alldredge@state.co.us Department of Fish, Wildlife, and Conservation Biology, Colorado State University 115 Wagar, Fort Collins, CO 80523, USA; kevin.crooks@colostate.edu Department of Molecular Diagnostics of Oncogenic Infections, Research Program Infection, Inflammation and Cancer, German Cancer Research Center, (Deutsches Krebsforschungszentrum Heidelberg, DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany; m.loechelt@dkfz-heidelberg.de School of Biological Sciences, University of Tasmania, Sandy Bay, Tasmania 7005, Australia; Scott.carver@utas.edu.au Correspondence: sue.vandewoude@colostate.edu Received: 7 March 2019; Accepted: 17 April 2019; Published: 19 April 2019 ���������� ������� Abstract: Feline foamy virus (FFV) is a retrovirus that has been detected in multiple feline species, including domestic cats (Felis catus) and pumas (Puma concolor). FFV results in persistent infection but is generally thought to be apathogenic. Sero-prevalence in domestic cat populations has been documented in several countries, but the extent of viral infections in nondomestic felids has not been reported. In this study, we screened sera from 348 individual pumas from Colorado, Southern California and Florida for FFV exposure by assessing sero-reactivity using an FFV anti-Gag ELISA. We documented a sero-prevalence of 78.6% across all sampled subpopulations, representing 69.1% in Southern California, 77.3% in Colorado, and 83.5% in Florida. Age was a significant risk factor for FFV infection when analyzing the combined populations. This high prevalence in geographically Viruses 2019, 11, 359; doi:10.3390/v11040359 www.mdpi.com/journal/viruses �Viruses 2019, 11, 359 2 of 9 distinct populations reveals widespread exposure of puma to FFV and suggests efficient shedding and transmission in wild populations. Keywords: feline foamy virus; epidemiology; retrovirus; Spumaretrovirus; mountain lion; Puma concolor; ELISA 1. Introduction Feline foamy virus (FFV) is a member of the oldest retrovirus family, Spumaretrovirinae [1]. The virus is reportedly contact-dependent and causes life-long infections in felines worldwide [2]. FFV was originally identified as a tissue culture contaminant from primary feline cell cultures [3] and named for its characteristic cytopathic effects. In comparison to other feline retroviruses, the relevance of FFV infection to felid behavior and health is not yet well-understood despite its high prevalence in populations worldwide [4]. Existing epidemiological studies on FFV have almost exclusively evaluated domestic cat (Felis catus) populations [5,6], with only a handful evaluating prevalence in wild feline species [7–10]. Additionally, published literature has found FFV to be putatively apathogenic in domestic cats [11] but no literature has explored the virus’ relationship to pathology in wild felids. Puma (Puma concolor) are the largest felid in North America and have frequent contact (e.g. predation) with domestic cats [12], making them a unique subject for this analysis. In this study we exploited an extensive archive [13] to investigate FFV sero-prevalence and risk factors in pumas in the United States using a serologic assay validated for use in domestic cats [14–16]. Relationships between puma demography (e.g. age and sex) and FFV infection was determined using a Bayesian hierarchical modeling approach. We additionally explored the effect of a regional treatment (sport-hunting ban) on FFV sero-positivity in one Colorado subpopulation in order to investigate the effect of management interventions on the spread of the virus. Our results suggested that FFV sero-prevalence in U.S. puma was high (78.6% overall), risk factors varied by sampling location, and that a ban on hunting did not affect FFV sero-prevalence in Colorado. 2. Materials and Methods We evaluated FFV sero-prevalence in three states: Colorado (n = 130, collected 2005–2011), Florida (n = 150, collected 1983–2010), and Southern California (n = 68, collected 2001–2011). Puma samples were opportunistically collected by government and local authorities engaged in independent management studies as previously described [13,17]. Sex and age of sampled pumas, if recorded, were determined via manager expertise, categorized as male or female and adult or young. Due to inconsistent location information, each state’s sample population was considered a uniform population. Sera were tested in duplicate on separate 96-well plates at 1:50 dilution using a non-quantitative GST-capture ELISA targeting the FFV Gag antigen, as previously described [14–16]. The ELISA has high sensitivity and specificity for the detection of FFV antibodies in naturally and experimentally infected domestic cats and has been validated against western blot [14–16]. This ELISA utilizes recombinant FFV Gag antigen generated from domestic cat FV sequences, which are 98% similar to the published puma FFV Gag [18,19]. Additional data evaluating more than 50 gag sequences from Colorado pumas has determined 95–100% similarity between puma and domestic cat FFVs [20]. A positive ELISA result was defined as having an OD absorbance over [2 × (meanGag + 3 SD)], with “meanGag” being the average negative control absorbance, and “SD” being the standard deviation of the negative control absorbance. This calculation employs very stringent cut off criteria [14–16], and is similar to methods of analytical detection reported by Lardeux et al. (2016) [21]. OD calculation was revised for each run, assuring each analysis was compared to its own negative control. Serum from experimentally infected domestic cats (positive) or specific pathogen free domestic cats (negative) were used as controls [15]. All plates were run in the same laboratory by the same individual over the course of one month. �Viruses 2019, 11, 359 3 of 9 Reagents were reconstituted on an as-needed basis. ELISA plates were prepared with fresh coating buffer the night prior to use. Normalization results was conducted by calculating each sample’s absorbance as a percent Viruses 2019, 11, x FOR of PEER REVIEW 3 of 9 of average positive control absorbance on the same plate. Each sample was additionally recorded as of results wasoff conducted by calculating each sample’s absorbance as a percent of positiveNormalization or negative using OD cut values calculated as described above. average positive control absorbance on the same plate. Each sample was additionally recorded FFV sero-prevalence across the sampling period, stratified by sex (male and female) andasage positive or negative using OD cut off values calculated as described above. (young and adult) across all locations was compared using a chi-square test. Sex, age, and the FFV sero-prevalence across the sampling period, stratified by sex (male and female) and age interaction between sex and age were evaluated as possible risk factors for FFV infection for each state (young and adult) across all locations was compared using a chi-square test. Sex, age, and the and across all samples using Bayesian generalized linear models (GLMs, a style of linear regression interaction between sex and age were evaluated as possible risk factors for FFV infection for each accounting for response variables with non-normal error distributions). For each coefficient (i.e., state and across all samples using Bayesian generalized linear models (GLMs, a style of linear variable), we used weakly informative priors and extracted a 95% credible interval from the posterior regression accounting for response variables with non-normal error distributions). For each distribution. Any coefficient whose 95% credible interval did not contain 0 was considered important. coefficient (i.e., variable), we used weakly informative priors and extracted a 95% credible interval GLMs were ranked comparedAny usingcoefficient Akaike information an did estimator of the0relative from the posteriorand distribution. whose 95%criterion credible (AIC, interval not contain was quality of a statistical model when compared to other models for Akaike a given information set of data). criterion The model with considered important. GLMs were ranked and compared using (AIC, thean lowest AIC value was considered to better fit the data and subsequently the important variable(s) estimator of the relative quality of a statistical model when compared to other models for a given within model considered riskvalue factorswas for considered puma FFV infection. If athe model set ofthat data). Thewere model with thepotential lowest AIC to better fit data had andan important predictor and was within 2 AIC of the best fit model, it was considered reveal the for most subsequently the important variable(s) within that model were considered potentialtorisk factors credible risk factor for FFV infection in pumas. Puma with unrecorded sex (n = 10) or age (n = puma FFV infection. If a model had an important predictor and was within 2 AIC of the best fit model,74) were excluded from to risk factorthe analyses. There risk werefactor 173 female, male, 190 adult and 86 with young it was considered reveal most credible for FFV167 infection in pumas. Puma unrecorded = 10) or age (n = 74) were excluded from risk factor analyses. There were 173 female, pumas used insex the(nanalysis. 167We male, 190 adult evaluated and 86 young used in the during analysis.and after a management intervention in additionally FFVpumas sero-prevalence We additionally evaluated FFV sero-prevalence during and implemented after a management intervention in in Colorado. Between 2004 and 2009, Colorado Parks and Wildlife a sport hunting ban Between 2004 andmanagement 2009, Colorado Parks and Wildlife implemented sport hunting ban in theColorado. Western Slope to evaluate programs [22]. We compared FFVasero-prevalence during the Western Slope to evaluate management programs [22]. We compared FFV sero-prevalence during (2004–2009) and after (2010–2014) the sport hunting ban using a chi square statistic. (2004–2009) and after (2010–2014) the sport hunting ban using a chi square statistic. 3. Results 3. Results 3.1. Sero-Prevalence 3.1. Sero-Prevalence FFV sero-prevalence is reported in Figure 1 and was high in all three states with an overall FFV sero-prevalence is reported in Figure and was high in allassociation three statesof with ansero-prevalence overall serosero-prevalence of 78.6% (95% CI: 74, 82.7). There1 was no significant FFV prevalence of 78.6% (95% CI: 74, 82.7). There was no significant association of FFV sero-prevalence with location at the state level. with location at the state level. 100 90 % Sero-prevalence 80 70 60 50 40 30 20 10 0 S. California (n=68) Colorado (n=130) Florida (n= 150) Overall (n=348) Figure 1. 1. Puma feline sero-prevalenceininthe theU.S. U.S.Sero-prevalence Sero-prevalence Figure Puma felinefoamy foamyvirus viruswas wasfound found in in high high sero-prevalence byby state: Southern Colorado77.3% 77.3%(95% (95%CI: CI:69.1, 69.1,84.3%); 84.3%); Florida state: SouthernCalifornia California69.1% 69.1%(95% (95%CI: CI: 56.7, 56.7, 79.8%); 79.8%); Colorado Florida 83.5% (95% CI:CI: 77.0, 88.9%). Sero-prevalence 83.5% (95% 77.0, 88.9%). Sero-prevalenceacross acrossallallsampling samplinglocations locations(overall) (overall)was was78.6% 78.6%(95% (95%CI: 74.0, ThereThere was no difference between states (p =(p0.14). CI:82.7%). 74.0, 82.7%). wassignificant no significant difference between states = 0.14). Figure 2 displays the normalized distribution of all samples used in this analysis, grouped by location, and colored by negative and positive assignment. Colorado samples with normalized �Viruses 2019, 11, 359 4 of 9 Figure 2 displays the normalized distribution of all samples used in this analysis, grouped Viruses 2019, 11, x FOR PEER REVIEW 4 of 9 by location, and colored by negative and positive assignment. Colorado samples with normalized absorbance >70% wereconsistently consistentlyclassified classifiedasaspositive, positive, while samples absorbance >70%ofofthe thepositive positive control control values values were while samples <70% ofof the positive SouthernCalifornia Californiasamples samples classified <70% the positivecontrol’s control’sabsorbance absorbance were were negative. negative. Southern classified as as positive had absorbance values >50% of the positive control absorbance except for one sample that positive had absorbance values >50% positive control absorbance except for one sample that was classified31% 31%ofofpositive positivecontrol control value value but still above AllAll other was classified above negative negativecut cutoff offfor forthe theplate. plate. other SouthernCalifornia Californiasamples samplesclassified classified as as negative negative had absorbance absorbance <50%. and Southern Southern <50%.Colorado Colorado and Southern California samples classified positivewere wereclearly clearlydistinguished distinguishedby bynegative negative cut cut off values (Figure 2). California samples classified asaspositive 2). While there was less demarcation of clearly negative and positive samples in thepopulation, Florida While there was less demarcation of clearly negative and positive samples in the Florida population, onlysamples two positive samples were <30% absorbance and all negative were <30%. only two positive were <30% absorbance and all negative samples were samples <30%. Reclassification Reclassification of all Florida panthers with an absorbance less than 49% (n = 7) as negative would in of all Florida panthers with an absorbance less than 49% (n = 7) as negative would have resulted have resulted in a sero-positivity of 74% which fell outside of the calculated 95% confidence interval a sero-positivity of 74% which fell outside of the calculated 95% confidence interval (estimated as (estimated as 77.0–88.9%); however, exclusion of these samples would have resulted in a sero77.0–88.9%); however, exclusion of these samples would have resulted in a sero-positivity of 78%, positivity of 78%, which remained within the calculated 95% confidence interval (estimated as 77.0– which remained within the calculated 95% confidence interval (estimated as 77.0–88.9%). 88.9%). % of Positive Absorbance (OD 450nm) 200 150 100 50 (n =6 8) A S. C (n =1 30 ) CO FL (n =1 50 ) 0 Figure 2. 2.Seropositive distinguishedfrom fromsamples samplesclassified classified seronegative. Figure SeropositiveOD ODvalues values were were well well distinguished as as seronegative. The sample points in red were negative; the sample points in green, purple or black were The sample points in red were negative; the sample points in green, purple or black were classified classified as positive for their respective locations. All All Colorado andand all but oneone Southern California sample fell into as positive for their respective locations. Colorado all but Southern California sample fell positive absorbance ranges ranges that were segregated (noted (noted with black classified into positive absorbance thatclearly were clearly segregated withlines) blackfrom lines)samples from samples as classified negative (<70% for CO,(<70% <50%for for CO, S.CA). 112 for of 119 Florida as seropositive were as negative <50% S.CA). 112samples of 119 classified Florida samples classified as at least 49% of positive OD of values, with only two samples (noted samples with black line). seropositive were atcontrol least 49% positive control OD positive values, with only <30% two positive <30% (noted with black line). 3.2. Demographic Associations 3.2.Results Demographic Associations of Bayesian GLMs are reported and graphically portrayed in Table 1 and Figure 3, respectively. sex nor age are were predictors FFV infection in Southern California, but there ResultsNeither of Bayesian GLMs reported andofgraphically portrayed in Table 1 and Figure 3, was a trend for Neither higher FFV exposure in adults relative to younger Age was a predictor for FFV respectively. sex nor age were predictors of FFV infectionpumas. in Southern California, but there infection over for all higher sites and also in Colorado andrelative Southern California as individual with for adult was a trend FFV exposure in adults to younger pumas. Age was asites, predictor FFV infection all sites also in Colorado and Southern California as was individual sites, with pumas being at over greater risk. and In Florida as an individual site, sex, not age, a predictor of FFV adult pumas at greater risk. In Florida infection, with being females being at greater risk. as an individual site, sex, not age, was a predictor of FFV infection, with females being at greater risk. �Viruses 2019, 11, 359 5 of 9 Viruses 2019, 11, x FOR PEER REVIEW 5 of 9 Sex 5 Age Overall Sex Age Model averaged coefficient 4 Florida Colorado 3 2 1 0 -1 California 0 0.2 0.4 0.6 0.8 Variable importance 1 1.2 -2 -3 California Colorado (A) Florida Overall (B) Figure 3. Older pumas and female Florida panthers at higher risk of feline foamy virus (FFV) infection. Figure 3. Older pumas and importance female Florida panthers at higher risk as ofrisk feline foamy virusshowing (FFV) (A) The plot displays variable weights for puma sex or age factors for FFV, infection. (A) The plot displays variable importance weights puma sex or age as risk factors for age to be more important in Colorado, Southern California andfor across the sampling locations (overall). FFV, showing age to be more important in Colorado, Southern California and across the sampling (B) The plot displays model averaged coefficients with 95% confidence intervals, with adult and male locations (overall). The plot displays model coefficients withdid 95%not confidence intervals, being >0, and young(B) and female being <0. If theaveraged 95% confidence interval cross 0, the variable with adult and male being >0, and young and female being <0. If the 95% confidence interval did not was considered important. Females were weakly associated with infection risk in Florida only. cross 0, the variable was considered important. Females were weakly associated with infection risk in Table 1. Age Florida only.alone as a risk factor for feline foamy virus (FFV) infection produced the best fit model in Southern California, Colorado and across all sampling locations (overall), while in Florida sex was the risk 1. factor the best model. Models from most to the least supported Table Age that aloneproduced as a risk factor forfitfeline foamy viruswere (FFV)ranked infection produced best fit modelby the in data. Southern California, Colorado and across all sampling locations (overall), while in Florida sex was the risk factor that produced the best fit model. Models were ranked from most to least supported by Model AIC ∆AIC Model Weight the data. SOUTHERN CALIFORNIA AIC Model Weight 0.46 Age Model 63.39 ΔAIC 0.00 Null 64.98 1.59 0.21 SOUTHERN Sex + Age 65.34 1.94 0.18 CALIFORNIA Sex 66.7 3.38 0.09 67.34 0.00 3.94 0.06 Sex + Age + Sex*Age 63.39 Age 0.46 COLORADO 1.59 64.98 Null Age 62.09 0.00 0.21 0.57 1.94 65.34 Sex + Age Sex + Age 63.87 1.78 0.18 0.23 66.7 Sex Sex + Age + Sex*Age 64.20 3.38 2.11 0.20 0.09 NullSex + Age + 77.69 15.60 0.00 3.94 67.34 Sex 79.23 17.14 0.06 0.00 Sex*Age FLORIDA COLORADO Sex 111.53 0.00 0.32 Sex + Age + Sex*Age 111.77 0.00 0.24 0.57 0.29 62.09 Age Sex + Age 112.55 1.02 0.19 63.87 1.78 0.23 Sex + Age Null 113.77 2.24 0.11 64.20 AgeSex + Age + 114.02 2.11 2.49 0.20 0.09 Sex*Age OVERALL Age Null Sex + Age Sex Sex + Age + Sex*Age Sex Null 77.69 15.60 0.00 240.22 0.00 79.23 17.14 0.00 240.84 0.62 242.83 2.61 254.40 14.18 256.12 15.90 0.50 0.37 0.14 0.00 0.00 �Viruses 2019, 11, 359 6 of 9 There was no significant difference in FFV sero-prevalence during (n = 40) and after (n = 85) the sport hunting ban implemented on the Western Slope of Colorado (Xˆ2: 2.5, p > 0.11). 4. Discussion This study provides the first broad scale investigation of FFV sero-prevalence in a large, geographically-dispersed sampling of free-ranging pumas. The high overall puma FFV sero-prevalence of 78.6% was higher than what had been recorded in domestic cat populations using similar ELISA assays [5,6]. Reported FFV infection rates in domestic cats have ranged from 30 to 70%, and have varied across locations and analytical methodology [2,5–7,15]. Although the puma sampling locations have marked differences in their landscapes and ecology [13] similarly high infection rates were detected in all populations. This suggests that FFV is readily transmitted across varied landscapes. While it was not possible to validate this study using sera from known positive and negative individuals, our stringency for negative and positive values was set using strict criteria [21]. Analysis of OD values illustrated distinct clustering of positive and negative sample absorbance (Figure 2), providing confidence that positive values truly reflected actual sero-prevalence. Florida samples were not as clearly distinguished as Colorado and Southern California samples. Samples from Florida were typically older and more likely to have been collected under less-than-ideal conditions from autolyzed animals (i.e., from road kill specimens). While this would typically result in the detection of more false negatives than false positives (due to degradation of circulating antibodies) it was also possible that there was higher nonspecific binding in this cohort. While exclusion of positive samples falling below 50% OD values of positive controls would still result in sero-prevalence estimates within the estimated 95% confidence interval in this population, further confirmatory analysis by PCR or other serologic assay would be warranted. The significant difference in sero-prevalence between age groups in Colorado and across the sampled locations suggests that adults have higher exposure to FFV. If adult pumas are at increased risk of FFV infection, it is necessary to further investigate FFV transmission and the potential for horizontal transmission between individuals being the dominant mode. The significantly lower sero-prevalence of FFV in kittens suggests that this age group does not have the same exposure to the virus as adults in the same populations, supporting horizontal transmission as the primary mode of transmission in these populations. We recognize the difficult task of sampling family units is needed to fully explore the possibility of vertical transmission of the virus; however, a future study with this goal could help to more clearly determine mother to cub transmission events. This reported high FFV sero-prevalence in adults exceeded the prevalence of most other infectious agents reported in these populations [13]. Adult pumas are traditionally considered to be solitary animals with rare documented instances of geographic and temporal overlap [23–25] though recent behavioral studies have exposed more social interactions between pumas [26]. In either case, inferred horizontal transmission of FFV resulting in four of five adults being exposed suggests facile transmission among adults. High FFV sero-prevalence could be explained by the virus maybe being able to survive on fomites (e.g., carcasses) long enough to infect other pumas without the need for physical contact between pumas. Further investigation of FFV degradation outside the host is needed to explore this alternative hypothesis. Further investigation of the virus’ genetic and biological attributes may provide unique insights into puma behavior and capacity for transmission of other pathogens similar to Fountain-Jones et al. (2018) [27]. The lack of significant difference in sero-prevalence between sex groups in Southern California, Colorado and overall is consistent with what we found in domestic cats [28]. This suggests transmission may be via non-antagonistic intraspecific interactions. Physical contacts during mating or saliva swapping during kill sharing may be the most likely types of primary transmission events, as there is no substantial evidence that prolonged amicable physical contact (e.g., grooming) is common between adults [23,24,26]. A recent report has documented higher FFV loads in saliva versus blood in naturally infected domestic cats [29], which could suggest a mechanism for puma to puma transmission. Further �Viruses 2019, 11, 359 7 of 9 studies of FFV ecology in the endangered and federally protected Florida panther (Puma concolor coryi) are needed to further our understanding of female sex as a risk factor for FFV infection. FFV has not been documented to cause acute disease or easily recognized clinical signs of disease in domestic cats [2,14,30]. However, simian Foamy Virus (SFV) has been associated with accelerated SIV disease [31] and an association between FFV viral load and exogenous Feline leukemia virus (FeLV) viremia has been noted [31,32]. Therefore, results from this study can be used to compare the prevalence of FFV to other retroviruses of pumas such as feline immunodeficiency virus (FIV) and evaluate possible potentiation of disease. Furthermore, since FFV is a lifelong infection and viral sequences can be isolated from circulating blood cells, genotypic analysis of puma FFV may be useful as a marker of animal movement and pathogen transmission within or between populations [33]. Author Contributions: Conceptualization, S.R.K., S.K., and S.V.; data curation, S.R.K.; formal analysis, S.R.K., N.D. and S.C.; funding acquisition, S.R.K., N.D., S.C. and S.V.; methodology, C.L.F. and M.L.; project administration, S.V.; resources, M.R.P., M.C., R.M., S.P.D.R., W.V., K.L., M.A. and K.C.; supervision, C.L.F., S.C. and S.V.; writing—original draft, S.R.K., N.D. and S.V.; writing—review and editing, S.R.K., N.D., S.K., C.L.F., J.M., M.C., S.P.D.R., W.V., K.L., M.A., K.C., M.L., S.C. and S.V. Funding: Funding and support were provided by Colorado State University, Colorado Parks and Wildlife (CPW), Boulder County Parks and Open Space, Boulder City Open Space and Mountain Parks, the Bureau of Land Management, U.S. Forest Service, Arizona State University, University of California at Davis, National Park Service, California State Parks, the Calabasas Landfill, The Santa Monica Mountains Fund, German Cancer Research Center, Boehringer Ingelheim, and a grant from the National Science Foundation-Ecology of Infectious Diseases Program (NSF EF-0723676; EF-1413925). Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Fields, B.N.; Knipe, D.M.; Howley, P.M.; Griffin, D.E. Foamy Viruses. In Fields Virology, 6th ed.; Rethwilm, A., Lindemann, D., Eds.; Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2013; p. 1614. Linial, M. Why aren’t foamy viruses pathogenic? Trends Microbiol. 2000, 8, 284–289. [CrossRef] Riggs, J.L.; Oshiro, L.S.; Taylor, D.O.N.; Lenette, E.H. Syncytium-forming agent isolated from domestic cats. Nature 1969, 11, 1190–1191. [CrossRef] Linial, M.L. Foamy Viruses are Unconventional Retroviruses. J. Virol. 1999, 73, 1747–1755. [PubMed] Lin, J.A.; Cheng, M.C.; Inoshima, Y.; Tomonaga, K.; Miyazawa, T.; Tohya, Y.; Toh, K.; Lu, Y.S.; Mikami, T. Seroepidemiological survey of feline retrovirus infections in cats in Taiwan in 1993 and 1994. J. Vet. Med. Sci. 1995, 57, 161–163. [CrossRef] [PubMed] Winkler, I.G.; Lochelt, M.; Flower, R.L.P. Epidemiology of Feline Foamy virus and Feline Immunodeficiency virus infections in domestic and feral cats: a seroepidemiological study. J. Clin. Microbiol. 1999, 37, 2848–2851. [PubMed] Mochizuki, M.; Akuzawa, M.; Nagatomo, H. Serological Survey of the Iriomote Cat (Felis-Iriomotensis) in Japan. J. Wildl. Dis. 1990, 26, 236–245. [CrossRef] [PubMed] Miyazawa, T.; Ikeda, Y.; Maeda, K.; Horimoto, T.; Tohya, Y.; Mochizuki, M.; Vu, D.; Vu, G.D.; Cu, D.X.; Ono, K.; et al. Seroepidemiological survey of feline retrovirus infections in domestic and leopard cats in northern Vietnam in 1997. J. Vet. Med. Sci. 1998, 60, 1273–1275. [CrossRef] Daniels, M.J.; Golder, M.C.; Jarrett, O.; MacDonald, D.W. Feline viruses in wildcats from Scotland. J. Wildl. Dis. 1999, 35, 121–124. [CrossRef] [PubMed] Cleaveland, S.; Mlengeya, T.; Kaare, M.; Haydon, D.; Lembo, T.; Luarenson, M.K.; Packer, C. The conservation relevance of epidemiological research into carnivore viral diseases in the Serengeti. Conservation Biol. 2007, 21, 612–622. [CrossRef] [PubMed] German, A.C.; Harbour, D.A.; Helps, C.R.; Gruffydd-Jones, T.J. Is feline foamy virus really apathogenic? Vet. Immunol. Immunopathol. 2008, 123, 114–118. [CrossRef] [PubMed] �Viruses 2019, 11, 359 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 8 of 9 Smith, J.; Wang, Y.; Wilmers, C. Spatial characteristics of residential development shift large carnivore prey habits. J. Wildl. Manage. 2016, 80, 1040–1048. [CrossRef] Carver, S.; Bevins, S.N.; Lappin, M.R.; Boydston, E.E.; Lyren, L.M.; Alldredge, M.; Logan, K.; Sweanor, L.L.; Riley, S.P.D.; Serieys, L.E.K.; et al. Pathogen exposure varies widely among sympatric populations of wild and domestic felids across the United States. Ecol. Appl. 2016, 26, 367–381. [CrossRef] Ledesma-Feliciano, C.; Hagen, S.; Troyer, R.; Zheng, X.; Musselman, E.; Slavkovic, L.D.; Franke, A.-M.; Maeda, D.; Zielonka, J.; Munk, C.; et al. Replacement of feline fomay virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential. Retrovirology 2018, 15, 38. [CrossRef] Bleiholder, A.; Muhle, M.; Hechler, T.; Bevins, S.; VandeWoude, S.; Denner, J.; Lochelt, M. Pattern of seroreactivity against feline foamy virus proteins in domestic cats from Germany. Vet. Immunol. Immunop. 2011, 143, 292–300. [CrossRef] Romen, F.; Pawlita, M.; Sehr, P.; Bachmann, S.; Schröder, J.; Lutz, H.; Löchelt, M. Antibodies against Gag are diagnostic markers for feline foamy virus infections while Env and Bet reactivity is undetectable in a substantial fraction of infected cats. Virology 2006, 345, 502–508. [CrossRef] [PubMed] Bevins, S.N.; Carver, S.; Boydston, E.E.; Lyren, L.M.; Alldredge, M.; Logan, K.A.; Riley, S.P.D.; Fisher, R.N.; Vickers, T.W.; Boyce, W.; et al. Three pathogens in sympatric populations of pumas, bobcats, and domestic cats: implications for infectious disease transmission. PLoS ONE 2006, 7, 31403. [CrossRef] [PubMed] Winkler, I.; Bodem, J.; Haas, L.; Zemba, M.; Delius, H.; Flower, R.; Flugel, R.M.; Löchelt, M. Characterization of the genome of feline foamy virus virus and its proteins shows distinct features different from those of primate spumaviruses. J. Virol. 1997, 71, 6727–6741. [PubMed] Kehl, T.; Bleiholder, A.; Roßmann, F.; Rupp, S.; Lei, J.; Lee, J.; Boyce, W.; Vickers, W.; Crooks, K.; VandeWoude, S. Complete Genome Sequences of Two Novel Puma concolor Foamy Viruses from California. Genome Announc. 2013, 1, e0020112. [CrossRef] [PubMed] Kraberger, S.; Arizona State University, Tempe, AZ, USA. Personal communication, 2019. Lardeux, F.; Torrico, G.; Aliaga, C. Calculation of ELISA’s cut-off based on the change-point analysis method for detection of Trypanosoma cruzi infection in Bolivian dogs in the absence of controls. Mem. Inst. Oswaldo Cruz. 2016, 111, 501–504. [CrossRef] [PubMed] Logan, K.A. Assessing Effects of Hunting on a Puma Population on the Uncompahgre Plateau, Colorado. Wildlife Research Report; Colorado Parks and Wildlife: Fort Collins, CO, USA, 2015. Seidensticker, J.; Hornocker, M.; Wiles, W.; Messick, J. Mountain Lion Social Organization in the Idaho Primitive Area. Wildl. Monogr. 1973, 35, 3–60. Hemker, T.; Lindzey, F.; Ackerman, B. Population Characteristics and Movement Patterns of Cougars in Southern Utah. J. Wildl. Manage. 1984, 48, 1275–1284. [CrossRef] Logan, K.A.; Sweanor, L. Behavior and social organization of a solitary carnivore. In Cougar: Ecology and Conservation; Hornocker, M., Negri, S., Eds.; University of Chicago Press: Chicago, IL, USA, 2010; pp. 105–117. Elbroch, M.; Levy, M.; Lubell, M.; Quigley, H.; Caragiulo, A. Adaptive social strategies in a solitary carnivore. Sci. Adv. 2017, 3, 1701218. [CrossRef] [PubMed] Fountain-Jones, N.M.; Pearse, W.D.; Escobar, L.E.; Alba-Casals, A.; Carver, S.; Davies, T.J.; Kraberger, S.; Papes, M.; Vandegrift, K.; Worsley-Tonks, K.; et al. Towards an eco-phylogenetic framework for infectious disease ecology. Biol. Reviews. 2017, 93, 950–970. [CrossRef] Kechejian, S.; Dannemiller, N.; Kraberger, S.; Ledesma-Feliciano, C.; Löchelt, M.; Carver, S.; VandeWoude, S. Feline foamy virus sero-prevalence and demographic risk factors in United States stray domestic cat populations. J. Feline Med. Surg. 2019. in revision. Cavalcante, L.T.F.; Muniz, C.P.; Jia, H.; Augusto, A.; Troccoli, F.; Medeiros, S.; Dias, C.; Switzer, W.; Soares, M.; Santos, A. Clinical and Molecular Features of Feline Foamy Virus and Feline Leukemia Virus Co-Infection in Naturally-Infected Cats. Viruses 2018, 10, 702. [CrossRef] [PubMed] Saib, A. Non-primate foamy viruses. Curr. Top. Microbiol. Immunol. 2003, 277, 197–211. [PubMed] Choudhary, A.; Galvin, T.A.; Williams, D.K.; Beren, J.; Bryant, M.A.; Khan, A.S. Influence of naturally occurring simian foamy viruses (SFVs) on SIV disease progression in the rhesus macaque (Macaca mulatta) model. Viruses 2013, 5, 1414–1430. [CrossRef] �Viruses 2019, 11, 359 32. 33. 9 of 9 Powers, J.A.; Chiu, E.S.; Kraberger, S.; Roelke-Parker, M.; Lowery, I.; Erbeck, K.; Troyer, R.; Carver, S.; VandeWoude, S. Feline leukemia virus disease outcomes in a domestic cat breeding colony: Relationship to endogenous FeLV and other chronic viral infections. J. Virol. 2018, 92, e00649-18. [CrossRef] Biek, R.; Real, L.A. The landscape genetics of infectious disease emergence and spread. Mol. Ecol. 2010, 19, 3515–3531. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Journal Articles Description An account of the resource CPW peer-reviewed journal publications Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Feline foamy virus is highly prevalent in free-ranging <em>puma concolor</em> from Colorado, Florida and Southern California Rights Information about rights held in and over the resource <a href="http://rightsstatements.org/vocab/InC-NC/1.0/" target="_blank" rel="noreferrer noopener">In Copyright - Non-Commercial Use Permitted</a> Date Created Date of creation of the resource. 2019-04-19 Subject The topic of the resource Feline foamy virus Epidemiology Retrovirus <em>Spumaretrovirus</em> Mountain lion <em>Puma concolor</em> ELISA Description An account of the resource <span>Feline foamy virus (FFV) is a retrovirus that has been detected in multiple feline species, including domestic cats (</span><span class="html-italic">Felis catus</span><span>) and pumas (</span><span class="html-italic">Puma concolor</span><span>). FFV results in persistent infection but is generally thought to be apathogenic. Sero-prevalence in domestic cat populations has been documented in several countries, but the extent of viral infections in nondomestic felids has not been reported. In this study, we screened sera from 348 individual pumas from Colorado, Southern California and Florida for FFV exposure by assessing sero-reactivity using an FFV anti-Gag ELISA. We documented a sero-prevalence of 78.6% across all sampled subpopulations, representing 69.1% in Southern California, 77.3% in Colorado, and 83.5% in Florida. Age was a significant risk factor for FFV infection when analyzing the combined populations. This high prevalence in geographically distinct populations reveals widespread exposure of puma to FFV and suggests efficient shedding and transmission in wild populations.</span> Creator An entity primarily responsible for making the resource Kechejian, Sarah R. Dannemiller, Nick Kraberger, Simona Ledesma-Feliciano, Carmen Malmberg, Jennifer Roelke Parker, Melody Cunningham, Mark McBride, Roy Riley, Seth P D Vickers, Winston T. Logan, Kenneth A. Alldredge, Mathew W. Crooks, Kevin R. Löchelt, Martin Carver, Scott VandeWoude, Sue Language A language of the resource English Is Part Of A related resource in which the described resource is physically or logically included. Viruses Format The file format, physical medium, or dimensions of the resource application/pdf Extent The size or duration of the resource. 9 pages Bibliographic Citation A bibliographic reference for the resource. Recommended practice is to include sufficient bibliographic detail to identify the resource as unambiguously as possible. Kechejian, S. R., N. Dannemiller, S. Kraberger, C. Ledesma-Feliciano, J. Malmberg, M. Roelke Parker, M. Cunningham, R. McBride, S. P. Riley, W. T. Vickers, K. Logan, M. Alldredge, K. Crooks, M. Löchelt, S. Carver, and S. VandeWoude. 2019. Feline foamy virus is highly prevalent in free-ranging <em>puma concolor</em> from Colorado, Florida and Southern California. Viruses 11:359. <a href="https://doi.org/10.3390/v11040359" target="_blank" rel="noreferrer noopener">https://doi.org/10.3390/v11040359</a> Type The nature or genre of the resource Article