Moderate Susceptibility to Subcutaneous Plague (Yersinia pestis) Challenge in Vaccine-Treated and Untreated Sonoran Deer Mice (Peromyscus maniculatus sonoriensis) and Northern Grasshopper Mice (Onychomys leucogaster).

The variable response of wild mice to Yersinia pestis infection, the causative agent of plague, has generated much speculation concerning their role in the ecology of this potentially lethal disease. Researchers have questioned the means by which Y. pestis is maintained in nature and also sought methods for managing the disease. Here we assessed the efficacy of a new tool, the sylvatic plague vaccine (SPV), in wild-caught northern grasshopper mice (Onychomys leucogaster) and commercially acquired Sonoran deer mice (Peromyscus maniculatus sonoriensis). More than 40% of the animals survived a subcutaneous Y. pestis challenge of 175,000 colony forming units (over 30,000 times the white mouse 50% lethal dose) in both vaccine-treated and control groups. Our results indicate that SPV distribution is unlikely to protect adult mice from plague infection in field settings and corroborate the heterogeneous response to Y. pestis infection in mice reported by others.

A Novel Retrovirus (Gunnison's Prairie Dog Retrovirus) Associated With Thymic Lymphoma in Gunnison's Prairie Dogs in Colorado, USA.

As part of research and wildlife disease surveillance efforts, we performed necropsy examinations of 125 free-ranging (n = 114) and captive (n = 11) prairie dogs in Colorado from 2009 to 2017. From these cases, we identified three cases of thymic lymphoma in free-ranging Gunnison's prairie dogs (Cynomys gunnisoni), and we identified a novel retroviral sequence associated with these tumors. The viral sequence is 7700 nucleotides in length and exhibits a genetic organization that is consistent with the characteristics of a type D betaretrovirus. The proposed name of this virus is Gunnison's prairie dog retrovirus (GPDRV). We screened all 125 prairie dogs for the presence of GPDRV using PCR with envelope-specific primers and DNA extracted from spleen samples. Samples were from Gunnison's prairie dogs (n = 59), black-tailed prairie dogs (Cynomys ludovicianus) (n = 40), and white-tailed prairie dogs (Cynomys leucurus) (n = 26). We identified GPDRV in a total of 7/125 (5.6%) samples including all three of the prairie dogs with thymic lymphoma, as well as spleen from an additional four Gunnison's prairie dogs with no tumors recognized at necropsy. None of the GPDRV-negative Gunnison's prairie dogs had thymic lymphomas. We also identified a related, apparently endogenous retroviral sequence in all prairie dog samples. These results suggest that GPDRV infection may lead to development of thymic lymphoma in Gunnison's prairie dogs.

Differential plague susceptibility in species and populations of prairie dogs.

Laboratory trials conducted over the past decade at U.S. Geological Survey National Wildlife Health Center indicate that wild populations of prairie dogs (Cynomys spp.) display different degrees of susceptibility to experimental challenge with fully virulent Yersinia pestis, the causative agent of plague. We evaluated patterns in prairie dog susceptibility to plague to determine whether the historical occurrence of plague at location of capture was related to survival times of prairie dogs challenged with Y. pestis. We found that black-tailed prairie dogs (Cynomys ludovicianus) from South Dakota (captured prior to the detection of plague in the state), Gunnison's prairie dogs (Cynomys gunnisoni) from Colorado, and Utah prairie dogs (Cynomys parvidens) from Utah were most susceptible to plague. Though the susceptibility of black-tailed prairie dogs in South Dakota compared with western locations supports our hypothesis regarding historical exposure, both Colorado and Utah prairie dogs have a long history of exposure to plague. It is possible that for these populations, genetic isolation/bottle necks have made them more susceptible to plague outbreaks.

Local factors associated with on-host flea distributions on prairie dog colonies.

Outbreaks of plague, a flea-vectored bacterial disease, occur periodically in prairie dog populations in the western United States. In order to understand the conditions that are conducive to plague outbreaks and potentially predict spatial and temporal variations in risk, it is important to understand the factors associated with flea abundance and distribution that may lead to plague outbreaks. We collected and identified 20,041 fleas from 6,542 individual prairie dogs of four different species over a 4-year period along a latitudinal gradient from Texas to Montana. We assessed local climate and other factors associated with flea prevalence and abundance, as well as the incidence of plague outbreaks. Oropsylla hirsuta, a prairie dog specialist flea, and Pulex simulans, a generalist flea species, were the most common fleas found on our pairs. High elevation pairs in Wyoming and Utah had distinct flea communities compared with the rest of the study pairs. The incidence of prairie dogs with Yersinia pestis detections in fleas was low (n = 64 prairie dogs with positive fleas out of 5,024 samples from 4,218 individual prairie dogs). The results of our regression models indicate that many factors are associated with the presence of fleas. In general, flea abundance (number of fleas on hosts) is higher during plague outbreaks, lower when prairie dogs are more abundant, and reaches peak levels when climate and weather variables are at intermediate levels. Changing climate conditions will likely affect aspects of both flea and host communities, including population densities and species composition, which may lead to changes in plague dynamics. Our results support the hypothesis that local conditions, including host, vector, and environmental factors, influence the likelihood of plague outbreaks, and that predicting changes to plague dynamics under climate change scenarios will have to consider both host and vector responses to local factors.

Factors Influencing Uptake of Sylvatic Plague Vaccine Baits by Prairie Dogs.

Sylvatic plague vaccine (SPV) is a virally vectored bait-delivered vaccine expressing Yersinia pestis antigens that can protect prairie dogs (Cynomys spp.) from plague and has potential utility as a management tool. In a large-scale 3-year field trial, SPV-laden baits containing the biomarker rhodamine B (used to determine bait consumption) were distributed annually at a rate of approximately 100-125 baits/hectare along transects at 58 plots encompassing the geographic ranges of four species of prairie dogs. We assessed site- and individual-level factors related to bait uptake in prairie dogs to determine which were associated with bait uptake rates. Overall bait uptake for 7820 prairie dogs sampled was 70% (95% C.I. 69.9-72.0). Factors influencing bait uptake rates by prairie dogs varied by species, however, in general, heavier animals had greater bait uptake rates. Vegetation quality and day of baiting influenced this relationship for black-tailed, Gunnison's, and Utah prairie dogs. For these species, baiting later in the season, when normalized difference vegetation indices (a measure of green vegetation density) are lower, improves bait uptake by smaller animals. Consideration of these factors can aid in the development of species-specific SPV baiting strategies that maximize bait uptake and subsequent immunization of prairie dogs against plague.

Burrow Dusting or Oral Vaccination Prevents Plague-Associated Prairie Dog Colony Collapse.

Plague impacts prairie dogs (Cynomys spp.), the endangered black-footed ferret (Mustela nigripes) and other sensitive wildlife species. We compared efficacy of prophylactic treatments (burrow dusting with deltamethrin or oral vaccination with recombinant "sylvatic plague vaccine" [RCN-F1/V307]) to placebo treatment in black-tailed prairie dog (C. ludovicianus) colonies. Between 2013 and 2015, we measured prairie dog apparent survival, burrow activity and flea abundance on triplicate plots ("blocks") receiving dust, vaccine or placebo treatment. Epizootic plague affected all three blocks but emerged asynchronously. Dust plots had fewer fleas per burrow (P < 0.0001), and prairie dogs captured on dust plots had fewer fleas (P < 0.0001) than those on vaccine or placebo plots. Burrow activity and prairie dog density declined sharply in placebo plots when epizootic plague emerged. Patterns in corresponding dust and vaccine plots were less consistent and appeared strongly influenced by timing of treatment applications relative to plague emergence. Deltamethrin or oral vaccination enhanced apparent survival within two blocks. Applying insecticide or vaccine prior to epizootic emergence blunted effects of plague on prairie dog survival and abundance, thereby preventing colony collapse. Successful plague mitigation will likely entail strategic combined uses of burrow dusting and oral vaccination within large colonies or colony complexes.

Sylvatic Plague Vaccine Partially Protects Prairie Dogs (Cynomys spp.) in Field Trials.

Sylvatic plague, caused by Yersinia pestis, frequently afflicts prairie dogs (Cynomys spp.), causing population declines and local extirpations. We tested the effectiveness of bait-delivered sylvatic plague vaccine (SPV) in prairie dog colonies on 29 paired placebo and treatment plots (1-59 ha in size; average 16.9 ha) in 7 western states from 2013 to 2015. We compared relative abundance (using catch per unit effort (CPUE) as an index) and apparent survival of prairie dogs on 26 of the 29 paired plots, 12 with confirmed or suspected plague (Y. pestis positive carcasses or fleas). Even though plague mortality occurred in prairie dogs on vaccine plots, SPV treatment had an overall positive effect on CPUE in all three years, regardless of plague status. Odds of capturing a unique animal were 1.10 (95% confidence interval [C.I.] 1.02-1.19) times higher per trap day on vaccine-treated plots than placebo plots in 2013, 1.47 (95% C.I. 1.41-1.52) times higher in 2014 and 1.19 (95% C.I. 1.13-1.25) times higher in 2015. On pairs where plague occurred, odds of apparent survival were 1.76 (95% Bayesian credible interval [B.C.I.] 1.28-2.43) times higher on vaccine plots than placebo plots for adults and 2.41 (95% B.C.I. 1.72-3.38) times higher for juveniles. Our results provide evidence that consumption of vaccine-laden baits can protect prairie dogs against plague; however, further evaluation and refinement are needed to optimize SPV use as a management tool.

Responses of Juvenile Black-tailed Prairie Dogs ( Cynomys ludovicianus ) to a Commercially Produced Oral Plague Vaccine Delivered at Two Doses.

We confirmed safety and immunogenicity of mass-produced vaccine baits carrying an experimental, commercial-source plague vaccine (RCN-F1/V307) expressing Yersinia pestis V and F1 antigens. Forty-five juvenile black-tailed prairie dogs ( Cynomys ludovicianus ) were randomly divided into three treatment groups (n=15 animals/group). Animals in the first group received one standard-dose vaccine bait (5×107 plaque-forming units [pfu]; STD). The second group received a lower-dose bait (1×107 pfu; LOW). In the third group, five animals received two standard-dose baits and 10 were left untreated but in contact. Two vaccine-treated and one untreated prairie dogs died during the study, but laboratory analyses ruled out vaccine involvement. Overall, 17 of 33 (52%; 95% confidence interval for binomial proportion [bCI] 34-69%) prairie dogs receiving vaccine-laden bait showed a positive anti-V antibody response on at least one sampling occasion after bait consumption, and eight (24%; bCI 11-42%) showed sustained antibody responses. The STD and LOW groups did not differ (P≥0.78) in their proportions of overall or sustained antibody responses after vaccine bait consumption. Serum from one of the nine (11%; bCI 0.3-48%) surviving untreated, in-contact prairie dogs also had detectable antibody on one sampling occasion. We did not observe any adverse effects related to oral vaccination.

Using Off-the-Shelf Technologies to Mass Manufacture Oral Vaccine Baits for Wildlife.

Technology and infrastructure costs can limit access to oral vaccination tools for wildlife disease control. We describe vaccine bait mass manufacturing employing off-the-shelf technologies. Our approach has helped advance scaling-up of plague vaccination campaigns, but components of this production system could be translated into other wildlife vaccination applications.

SEASON OF DELTAMETHRIN APPLICATION AFFECTS FLEA AND PLAGUE CONTROL IN WHITE-TAILED PRAIRIE DOG (CYNOMYS LEUCURUS) COLONIES, COLORADO, USA.

In 2008 and 2009, we evaluated the duration of prophylactic deltamethrin treatments in white-tailed prairie dog ( Cynomys leucurus ) colonies and compared effects of autumn or spring dust application in suppressing flea numbers and plague. Plague occurred before and during our experiment. Overall, flea abundance tended to increase from May or June to September, but it was affected by deltamethrin treatment and plague dynamics. Success in trapping prairie dogs (animals caught/trap days) declined between June and September at all study sites. However, by September trap success on dusted sites (19%; 95% confidence interval [CI] 16-22%) was about 15-fold greater than on undusted control sites (1%; CI 0.3-4%; P≤0.0001). Applying deltamethrin dust as early as 12 mo prior seemed to afford some protection to prairie dogs. Our data showed that dusting even a portion of a prairie dog colony can prolong its persistence despite epizootic plague. Autumn dusting may offer advantages over spring in suppressing overwinter or early-spring flea activity, but timing should be adjusted to precede the annual decline in aboveground activity for hibernating prairie dog species. Large colony complexes or collections of occupied but fragmented habitat may benefit from dusting some sites in spring and others in autumn to maximize flea suppression in a portion of the complex or habitat year-round.

Ecological Traits Driving the Outbreaks and Emergence of Zoonotic Pathogens.

Infectious diseases that are transmitted from wildlife hosts to humans, such as the Ebola virus and MERS virus, can be difficult to understand because the pathogens emerge from complex multifaceted ecological interactions. We use a wildlife-pathogen system-prairie dogs (Cynomys ludovicianus) and the plague bacterium (Yersinia pestis)-to describe aspects of disease ecology that apply to many cases of emerging infectious disease. We show that the monitoring and surveillance of hosts and vectors during the buildup to disease outbreaks are crucial for understanding pathogen-transmission dynamics and that a community-ecology framework is important to identify reservoir hosts. Incorporating multidisciplinary approaches and frameworks may improve wildlife-pathogen surveillance and our understanding of seemingly sporadic and rare pathogen outbreaks.

Apparent field safety of a raccoon poxvirus-vectored plague vaccine in free-ranging prairie dogs (Cynomys spp.), Colorado, USA.

Prairie dogs (Cynomys spp.) suffer high rates of mortality from plague. An oral sylvatic plague vaccine using the raccoon poxvirus vector (designated RCN-F1/V307) has been developed for prairie dogs. This vaccine is incorporated into palatable bait along with rhodamine B as a biomarker. We conducted trials in August and September 2012 to demonstrate uptake and apparent safety of the RCN-F1/V307 vaccine in two prairie dog species under field conditions. Free-ranging prairie dogs and other associated small rodents readily consumed vaccine-laden baits during field trials with no apparent adverse effects; most sampled prairie dogs (90%) and associated small rodents (78%) had consumed baits. Visual counts of prairie dogs and their burrows revealed no evidence of prairie dog decline after vaccine exposure. No vaccine-related morbidity, mortality, or gross or microscopic lesions were observed. Poxviruses were not isolated from any animal sampled prior to bait distribution or on sites that received placebo baits. We isolated RCN-F1/V307 from 17 prairie dogs and two deer mice (Peromyscus maniculatus) captured on sites where vaccine-laden baits were distributed. Based on these findings, studies examining the utility and effectiveness of oral vaccination to prevent plague-induced mortality in prairie dogs and associated species are underway.

Season and application rates affect vaccine bait consumption by prairie dogs in Colorado and Utah, USA.

Plague, a zoonotic disease caused by the bacterium Yersinia pestis, causes high rates of mortality in prairie dogs (Cynomys spp.). An oral vaccine against plague has been developed for prairie dogs along with a palatable bait to deliver vaccine and a biomarker to track bait consumption. We conducted field trials between September 2009 and September 2012 to develop recommendations for bait distribution to deliver plague vaccine to prairie dogs. The objectives were to evaluate the use of the biomarker, rhodamine B, in field settings to compare bait distribution strategies, to compare uptake of baits distributed at different densities, to assess seasonal effects on bait uptake, and to measure bait uptake by nontarget small mammal species. Rhodamine B effectively marked prairie dogs' whiskers during these field trials. To compare bait distribution strategies, we applied baits around active burrows or along transects at densities of 32, 65, and 130 baits/ha. Distributing baits at active burrows or by transect did not affect uptake by prairie dogs. Distributing baits at rates of ≥ 65/ha (or ≥ 1 bait/active burrow) produced optimal uptake, and bait uptake by prairie dogs in the autumn was superior to uptake in the spring. Six other species of small mammals consumed baits during these trials. All four species of tested prairie dogs readily consumed the baits, demonstrating that vaccine uptake will not be an obstacle to plague control via oral vaccination.

Duration of plague (Yersinia pestis) outbreaks in black-tailed prairie dog (Cynomys ludovicianus) colonies of northern Colorado.

Plague, caused by the bacterium Yersinia pestis, triggers die-offs in colonies of black-tailed prairie dogs (Cynomys ludovicianus), but the time-frame of plague activity is not well understood. We document plague activity in fleas from prairie dogs and their burrows on three prairie dog colonies that suffered die-offs. We demonstrate that Y. pestis transmission occurs over periods from several months to over a year in prairie dog populations before observed die-offs.

Flea abundance on black-tailed prairie dogs (Cynomys ludovicianus) increases during plague epizootics.

Black-tailed prairie dogs (Cynomys ludovicianus) on the Great Plains of the United States are highly susceptible to plague, caused by the bacterium Yersinia pestis, with mortality on towns during plague epizootics often approaching 100%. The ability of flea-borne transmission to sustain disease spread has been questioned because of inefficiency of flea vectors. However, even with low individual efficiency, overall transmission can be increased if flea abundance (the number of fleas on hosts) increases. Changes in flea abundance on hosts during plague outbreaks were recorded during a large-scale study of plague outbreaks in prairie dogs in north central Colorado during 3 years (2004-2007). Fleas were collected from live-trapped black-tailed prairie dogs before and during plague epizootics and tested by PCR for the presence of Y. pestis. The predominant fleas were two prairie dog specialists (Oropsylla hirsuta and Oropsylla tuberculata cynomuris), and a generalist flea species (Pulex simulans) was also recorded from numerous mammals in the area. The three species differ in seasonal abundance, with greatest abundance in spring (February and March) and fall (September and October). Flea abundance and infestation intensity increased during epizootics and were highest on prairie dogs with Y. pestis-infected fleas. Seasonal occurrence of epizootics among black-tailed prairie dogs was found to coincide with seasonal peaks in flea abundance. Concentration of infected fleas on surviving animals may account for rapid spread of plague during epizootics. In particular, the role of the generalist flea P. simulans was previously underappreciated.

Evidence for the involvement of an alternate rodent host in the dynamics of introduced plague in prairie dogs.

1. The introduction of plague to North America is a significant threat to colonies of prairie dogs (Cynomys ludovicianus), a species of conservation concern in the Great Plains. Other small rodents are exposed to the causative agent, Yersinia pestis, during or after epizootics; yet, its effect on these rodents is not known, and their role in transmitting and maintaining plague in the absence of prairie dogs remains unclear. 2. We live-trapped small rodents and collected their fleas on 11 colonies before, during and after plague epizootics in Colorado, USA, from 2004 to 2006. Molecular genetic (polymerase chain reaction) assays were used to identify Y. pestis in fleas. 3. Abundance of northern grasshopper mice (Onychomys leucogaster) was low on sites following epizootics in 2004, and declined markedly following plague onset on other colonies in 2005. These changes coincided with exposure of grasshopper mice to plague, and with periods when mice became infested with large numbers of prairie dog fleas (Oropsylla hirsuta), including some that were infected with Y. pestis. Additionally, several Pleochaetis exilis, fleas restricted to grasshopper mice and never found on prairie dogs on our site, were polymerase chain reaction-positive for Y. pestis, indicating that grasshopper mice can infect their own fleas. No changes in abundance of other rodent species could be attributed to plague, and no other rodents hosted O. hirsuta during epizootics, or harboured Y. pestis-infected fleas. 4. In spring 2004, grasshopper mice were most numerous in colonies that suffered plague the following year, and the pattern of colony extinctions over a 12-year period mirrored patterns of grasshopper mouse abundance in our study area, suggesting that colonies with high densities of grasshopper mice may be more susceptible to outbreaks. We speculate that grasshopper mice help spread Y. pestis during epizootics through their ability to survive infection, harbour prairie dog fleas and, during their wide-ranging movements, transport infected fleas among burrows, which functionally connects prairie dog coteries that would otherwise be socially distinct.

Estimating abundance using mark-resight when sampling is with replacement or the number of marked individuals is unknown.

Although mark-resight methods can often be a less expensive and less invasive means for estimating abundance in long-term population monitoring programs, two major limitations of the estimators are that they typically require sampling without replacement and/or the number of marked individuals available for resighting to be known exactly. These requirements can often be difficult to achieve. Here we address these limitations by introducing the Poisson log and zero-truncated Poisson log-normal mixed effects models (PNE and ZPNE, respectively). The generalized framework of the models allow the efficient use of covariates in modeling resighting rate and individual heterogeneity parameters, information-theoretic model selection and multimodel inference, and the incorporation of individually unidentified marks. Both models may be implemented using standard statistical computing software, but they have also been added to the mark-recapture freeware package Program MARK. We demonstrate the use and advantages of (Z)PNE using black-tailed prairie dog data recently collected in Colorado. We also investigate the expected relative performance of the models in simulation experiments. Compared to other available estimators, we generally found (Z)PNE to be more precise with little or no loss in confidence interval coverage. With the recent introduction of the logit-normal mixed effects model and (Z)PNE, a more flexible and efficient framework for mark-resight abundance estimation is now available for the sampling conditions most commonly encountered in these studies.

Transmission efficiency of two flea species (Oropsylla tuberculata cynomuris and Oropsylla hirsuta) involved in plague epizootics among prairie dogs.

Plague, caused by Yersinia pestis, is an exotic disease in North America circulating predominantly in wild populations of rodents and their fleas. Black-tailed prairie dogs (Cynomys ludovicianus) are highly susceptible to infection, often experiencing mortality of nearly all individuals in a town as a result of plague. The fleas of black-tailed prairie dogs are Oropsylla tuberculata cynomuris and Oropsylla hirsuta. We tested the efficiency of O. tuberculata cynomuris to transmit Y. pestis daily from 24 to 96 h postinfection and compared it to previously collected data for O. hirsuta. We found that O. tuberculata cynomuris has over threefold greater transmission efficiency (0.18 infected fleas transmit Y. pestis at 24 h postinfection) than O. hirsuta (0.05 fleas transmit). Using a simple model of flea-borne transmission, we combine these laboratory measurements with field data on monthly flea loads to compare the seasonal vectorial capacity of these two flea species. Coinciding with seasonal patterns of flea abundance, we find a peak in potential for flea-borne transmission in March, during high O. tuberculata cynomuris abundance, and in September-October when O. hirsuta is common. Our findings may be useful in determining the timing of insecticidal dusting to slow plague transmission in black-tailed prairie dogs.

Exposure of small rodents to plague during epizootics in black-tailed prairie dogs.

Plague, caused by the bacterium Yersinia pestis, causes die-offs of colonies of prairie dogs (Cynomys ludovicianus). It has been argued that other small rodents are reservoirs for plague, spreading disease during epizootics and maintaining the pathogen in the absence of prairie dogs; yet there is little empirical support for distinct enzootic and epizootic cycles. Between 2004 and 2006, we collected blood from small rodents captured in colonies in northern Colorado before, during, and for up to 2 yr after prairie dog epizootics. We screened 1,603 blood samples for antibodies to Y. pestis, using passive hemagglutination and inhibition tests, and for a subset of samples we cultured blood for the bacterium itself. Of the four species of rodents that were common in colonies, the northern grasshopper mouse (Onychomys leucogaster) was the only species with consistent evidence of plague infection during epizootics, with 11.1-23.1% of mice seropositive for antibody to Y. pestis during these events. Seropositive grasshopper mice, thirteen-lined ground squirrels (Spermophilus tridecemlineatus), and deer mice (Peromyscus maniculatus) were captured the year following epizootics. The appearance of antibodies to Y. pestis in grasshopper mice coincided with periods of high prairie dog mortality; subsequently, antibody prevalence rates declined, with no seropositive individuals captured 2 yr after epizootics. We did not detect plague in any rodents off of colonies, or on colonies prior to epizootics, and found no evidence of persistent Y. pestis infection in blood cultures. Our results suggest that grasshopper mice could be involved in epizootic spread of Y. pestis, and possibly, serve as a short-term reservoir for plague, but provide no evidence that the grasshopper mouse or any small rodent acts as a long-term, enzootic host for Y. pestis in prairie dog colonies.

The potential role of swift foxes (Vulpes velox) and their fleas in plague outbreaks in prairie dogs.

Swift foxes (Vulpes velox) have been proposed as potential carriers of fleas infected with the bacterium Yersinia pestis between areas of epizootics in black-tailed prairie dogs (Cynomys ludovicianus). We examined antibody prevalence rates of a population of swift foxes in Colorado, USA, and used polymerase chain reaction (PCR) assays to examine their flea biota for evidence of Y. pestis. Fifteen of 61 (24%) captured foxes were seropositive, and antibody prevalence was spatially correlated with epizootic plague activity in prairie dog colonies in the year of, and previous to, the study. Foxes commonly harbored the flea Pulex simulans, though none of the fleas was positive for Y. pestis.