A plague epizootic in the white-tailed prairie dogs (Cynomys leucurus) of Meeteetse, Wyoming

Surveillance for sylvatic plague (Yersinia pestis) was conducted near Meeteetse, Wyoming (USA) from 24 May to 14 June 1985. Ten species of fleas were collected from white-tailed prairie dogs (Cynomys leucurus), and from their burrows and associated rodents. Five of these flea species and two adult prairie dogs were positive for plague. The progression of this plague epizootic appeared to be slower and the intensity was less than in previous epizootics in other prairie dog colonies. The plague epizootic occurred within the only known colony of black-footed ferrets (Mustela nigripes) and was a potential threat to the food source of this endangered species.     

Canine distemper in black-footed ferrets (Mustela nigripes) from Wyoming

In September and October 1985, six black-footed ferrets (Mustela nigripes) were captured from the only known population, located near Meeteetse, Wyoming for captive propagation. Two days following capture an adult male showed signs of canine distemper and an adult female displayed similar signs 7 days postcapture; these infections were undoubtedly acquired prior to capture. Subsequently the four remaining captive black-footed ferrets also developed canine distemper and all eventually died. Clinical signs included severe pruritus, hyperkeratosis and progressive loss of body condition. A few animals had intermittent diarrhea and respiratory disease. Intranuclear and intracytoplasmic inclusion bodies were numerous in epithelial tissues and two black-footed ferrets had a mild to moderate meningoencephalitis. Canine distemper virus was isolated from four animals and paramyxovirus nucleocapsids were observed by electron microscopy of feces from all affected black-footed ferrets. Antibodies to canine distemper virus were not detected in sera of sick black-footed ferrets. Antibodies to canine distemper virus were found in sera of badgers (Taxidea taxus) and coyotes (Canis latrans) collected in the Meeteetse area in 1986. Most free-ranging black-footed ferrets in the colony apparently died of canine distemper during the summer and fall of 1985. An attempt was made to capture all surviving animals in the affected area in order to abort the epizootic and provide black-footed ferrets for captive propagation.     

Susceptibility of the Siberian polecat to subcutaneous and oral Yersinia pestis exposure.

To determine if the Siberian polecat (Mustela eversmannii) represents a suitable model for the study of plague pathogenesis and prevention in the black-footed ferret (Mustela nigripes), polecats were exposed to 10(3), 10(7), or 10(10) Yersinia pestis organisms by subcutaneous injection; an additional group was exposed to Y. pestis via ingestion of a plague-killed mouse. Plague killed 88% of polecats exposed to Y. pestis (71% mortality in the 10(3) group, 100% mortality in the 10(7) and 10(10) groups, and 83% mortality in the mouse-fed group). Within the challenged group, mean day of death post-challenge ranged from 3.6 to 7.6 days; all polecats died on or before day 12 post-challenge. Animals receiving the lowest parenteral dose survived significantly longer than those receiving higher parenteral doses. Within challenged animals, mean survival time was lower in those presenting with significant weight loss by day 3, lethargy, and low fecal output; time to onset of lethargy and other signs was also related to risk of dying and/or plague dose. Six polecats developed serum antibody titers to the Y. pestis F1 protein. Three seropositive polecats survived the initial challenge and a subsequent exposure to a plague-killed mouse, while two seropositive animals later died. This study confirms that the Siberian polecat is susceptible to plague and suggests that this species will offer an appropriate surrogate for black-footed ferrets in future plague studies and related vaccine trials.     

Vaccination with F1-V fusion protein protects black-footed ferrets (Mustela nigripes) against plague upon oral challenge with Yersinia pestis

Previous studies have established that vaccination of black-footed ferrets (Mustela nigripes) with F1-V fusion protein by subcutaneous (SC) injection protects the animals against plague upon injection of the bacterium Yersinia pestis. This study demonstrates that the F1-V antigen can also protect ferrets against plague contracted via ingestion of a Y. pestis-infected mouse, a probable route for natural infection. Eight black-footed ferret kits were vaccinated with F1-V protein by SC injection at approximately 60 days-of-age. A booster vaccination was administered 3 mo later via SC injection. Four additional ferret kits received placebos. The animals were challenged 6 wk after the boost by feeding each one a Y. pestis-infected mouse. All eight vaccinates survived challenge, while the four controls succumbed to plague within 3 days after exposure. To determine the duration of antibody postvaccination, 18 additional black-footed ferret kits were vaccinated and boosted with F1-V by SC injection at 60 and 120 days-of-age. High titers to both F1 and V (mean reciprocal titers of 18,552 and 99,862, respectively) were found in all vaccinates up to 2 yr postvaccination, whereas seven control animals remained antibody negative throughout the same time period.     

A panel of microsatellite markers for genetic studies of European polecats (Mustela putorius) and ferrets (Mustela furo)

We report a panel of 12 microsatellite markers, including nine novel polymorphic loci isolated in the European polecat and three loci previously developed in closely related species, for genetic studies of polecats and ferrets. We tested the panel at fine geographic scales in polecat and domestic ferret populations of Britain and Portugal and at a broad geographic scale, using 50 polecat samples from across Western Europe. At the fine geographic scales, we recorded one to five alleles per locus and observed and expected heterozygosities ranging from 0 to 0.80 and from 0 to 0.74, respectively. These values increased markedly in the European sample of polecats. No linkage disequilibrium was detected between any pair of loci and all genotypic frequencies complied with Hardy–Weinberg equilibrium expectations. The microsatellite panel allowed the molecular discrimination of polecats and domestic ferrets in Britain, where hybridisation between the two species has been documented. Thus, these loci may be useful not only in genetic studies of polecats and ferrets but also in studies to assess hybridisation between these two mustelid species.     

Prevalence of Yersinia pestis in rodents and fleas associated with black-tailed prairie dogs (Cynomys ludovicianus) at Thunder Basin National Grassland, Wyoming

Rodents (and their fleas) that are associated with prairie dogs are considered important for the maintenance and transmission of the bacterium (Yersinia pestis) that causes plague. Our goal was to identify rodent and flea species that were potentially involved in a plague epizootic in black-tailed prairie dogs at Thunder Basin National Grassland. We collected blood samples and ectoparasites from rodents trapped at off- and on-colony grids at Thunder Basin National Grassland between 2002 and 2004. Blood samples were tested for antibodies to Y. pestis F-1 antigen by a passive hemagglutination assay, and fleas were tested by a multiplex polymerase chain reaction, for the presence of the plague bacterium. Only one of 1,421 fleas, an Oropsylla hirsuta collected in 2002 from a deer mouse, Peromyscus maniculatus, tested positive for Y. pestis. Blood samples collected in summer 2004 from two northern grasshopper mice, Onychomys leucogaster, tested positive for Y. pestis antibodies. All three positive samples were collected from on-colony grids shortly after a plague epizootic occurred. This study confirms that plague is difficult to detect in rodents and fleas associated with prairie dog colonies, unless samples are collected immediately after a prairie dog die-off.     

Comparison of black-footed ferret (Mustela nigripes) and domestic ferret (M. putorius furo) courtship activity

Because of the scarcity of the endangered black-footed ferret (Mustela nigripes) and the amount of knowledge necessary for their conservation, surrogate research can play an important role in recovery. In this paper, we investigate surrogate behavioral research potential by comparing courtship behavior of the black-footed ferret to the congeneric domestic ferret (M. putorius furo). Ten female domestic ferrets were bred to five male domestic ferrets and eight female black-footed ferrets were bred to five black-footed ferret males. Courtship activities were defined, analyzed, and quantitatively compared between both groups. Lag sequential analysis of was used to prepare the behavioral matrices, and matrix cells were compared between groups with an equality of proportions test. Courtship patterns did not differ significantly between the two closely related species, and the domestic ferret would probably be an adequate surrogate for reproductive behavior research on the black-footed ferret.     

Toxoplasma gondii infections in captive black-footed ferrets (Mustela nigripes), 1992-1998: clinical signs, serology, pathology, and prevention

An epizootic of toxoplasmosis occurred among 22 adult and 30 kit black-footed ferrets (Mustela nigripes) maintained under quarantine conditions at the Louisville Zoological Garden (Louisville, Kentucky, USA) in June, 1992. Black-footed ferrets appear to be highly susceptible to acute and chronic toxoplasmosis. Clinical signs were observed in 19 adults and six kits and included anorexia, lethargy, corneal edema, and ataxia. Two adults and six kits died with acute disease. High antibody titers to Toxoplasma gondii were detected by latex agglutination and modified agglutination assay in 10 black-footed ferrets. One adult and six kits that died with acute clinical signs were necropsied and T. gondii-like organisms were found microscopically in multiple organs. Diagnosis of toxoplasmosis was confirmed by immunohistochemical staining with anti-T. gondii antibodies and by ultrastructural examination. Although the source of T. gondii for black-footed ferrets was not identified, frozen uncooked rabbit was the most likely source. Chronic toxoplasmosis resulted in the death of at additional 13 black-footed ferrets that were adults during the epizootic. Affected animals developed chronic progressive posterior weakness and posterior ataxia 6-69 mo after the epizootic began. Meningoencephalitis or meningoencephalomyelitis associated with chronic toxoplasmosis were identified at necropsy in all 13 ferrets. Precautions to prevent introduction of pathogens into the colony were insufficient to exclude T. gondii. Although toxoplasmosis may cause significant mortality in mustelids, the high mortality of black-footed ferrets in this epizootic was of concern due to their endangered status. This is the first detailed report of toxoplasmosis in black-footed ferrets.     

Black-footed ferret (Mustela nigripes) behavioral development: aboveground activity and juvenile play

We studied the behavioral development of black-footed ferrets (Mustela nigripes) from 6 to 16 post-natal weeks. At 6 weeks of age, kits are still lactating and depend on their mother for survival, while at 16 weeks, ferret young start achieving independence from their mother and littermates. Behavioral observations were obtained by placing videomonitors in the litters’ cages, nest boxes and in outdoor naturalistic enclosures. Captive-raised black-footed ferrets displayed nocturnal activity patterns, although they tended to appear aboveground at certain daytime hours presumably influenced by the established feeding and cleaning regimes. Growing ferrets began emerging aboveground at approximately 7 post-natal weeks and diel activity steadily increased as kits matured. The most manifest behavioral changes (appearance of new motor patterns, increase in aboveground play and in neck-biting behaviors) occurred from post-natal week 8 to week 12. This coincides with the period of maximum growth for ferrets and with a sensitive phase for the development of food preferences in this species. Changes from the 12 to the 16 post-natal weeks involved an increase in aboveground activity, including a higher frequency of scent-marking behaviors. Information provided in this study has important implications for enhancing the captive management of this endangered carnivore.     

Assessment of a recombinant F1-V fusion protein vaccine intended to protect Canada lynx (Lynx canadensis) from plague

As part of an ongoing restoration program in Colorado, USA, we evaluated adverse reactions and seroconversion in captive Canada lynx (Lynx canadensis) after vaccination with a recombinant F1-V fusion protein vaccine against Yersinia pestis, the bacterium that causes plague. Ten adult female lynx received the F1-V vaccine; 10 source- and age-matched lynx remained unvaccinated as controls. All of the vaccinated and control lynx remained apparently healthy throughout the confinement period. We observed no evidence of injection site or systemic reactions to the F1-V vaccine. Among vaccinated lynx, differences in log(10) reciprocal antibody titers measured in sera collected before and after vaccination (two doses) ranged from 1.2 to 5.2 for anti-F1 antibodies and from 0.6 to 5.2 for anti-V antibodies; titers in unvaccinated lynx did not change appreciably over the course of confinement prior to release, and thus differences in anti-F1 (P=0.003) and anti-V (P=0.0005) titers were greater among vaccinated lynx than among controls. Although our findings suggest that the F1-V fusion protein vaccine evaluated here is likely to stimulate antibody responses that may help protect Canada lynx from plague, we observed no apparent differences in survival between vaccinated and unvaccinated subject animals. Retrospectively, 22 of 50 (44%; 95% confidence interval 29-59%) unvaccinated lynx captured or recaptured in Colorado during 2000-08 had passive hemagglutination antibody titers >1:16, consistent with exposure to Y. pestis; paired pre- and postrelease titers available for eight of these animals showed titer increases similar in magnitude to those seen in response to vaccination, suggesting at least some lynx may naturally acquire immunity to plague in Colorado habitats.     

Use of protein microarray to identify gene expression changes of Yersinia pestis at different temperatures

Yersinia pestis is a bacterium that is transmitted between fleas, which have a body temperature of 26 °C, and mammalian hosts, which have a body temperature of 37 °C. To adapt to the temperature shift, phenotype variations, including virulence, occur. In this study, an antigen microarray including 218 proteins of Y. pestis was used to evaluate antibody responses in a pooled plague serum that was unadsorbed, adsorbed by Y. pestis cultivated at 26 °C, or adsorbed by Y. pestis cultivated at 26 and 37 °C to identify protein expression changes during the temperature shift. We identified 12 proteins as being expressed at 37 °C but not at 26 °C, or expressed at significantly higher levels at 37 °C than at 26 °C. The antibodies against 7 proteins in the serum adsorbed by Y. pestis cultivated at 26 and 37 °C remained positive, suggesting that they were not expressed on the surface of Y. pestis in LB broth in vitro or specifically expressed in vivo. This study proved that protein microarray and antibody profiling comprise a promising technique for monitoring gene expression at the protein level and for better understanding pathogenicity, to find new vaccine targets against plague.     

Genetic diversity and fitness in black-footed ferrets before and during a bottleneck

The black-footed ferret (Mustela nigripes) is an endangered North American carnivore that underwent a well-documented population bottleneck in the mid-1980s. To better understand the effects of a bottleneck on a free-ranging carnivore population, we used 24 microsatellite loci to compare genetic diversity before versus during the bottleneck, and compare the last wild population to two historical populations. We also compared genetic diversity in black-footed ferrets to that of two sibling species, the steppe polecat (Mustela eversmanni) and the European polecat (Mustela putorius). Black-footed ferrets during the bottleneck had less genetic diversity than steppe polecats. The three black-footed ferret populations were well differentiated (F(ST) = 0.57 +/- 0.15; mean +/- SE). We attributed the decrease in genetic diversity in black-footed ferrets to localized extinction of these genetically distinct subpopulations and to the bottleneck in the surviving subpopulation. Although genetic diversity decreased, female fecundity and juvenile survival were not affected by the population bottleneck.     

Effects of diet on captive black-footed ferret (Mustela nigripes) food preference

Black-footed ferrets (Mustela nigripes) are both habitat and prey specialists that depend on prairie dogs (Cynomys spp.) for food and utilize prairie dog burrows for refuge. In this study we investigated the effects of captive diet during early development on adult black-footed ferret food preferences. To test the hypothesis that early diet affects the food preferences of adult black-footed ferrets, we exposed 22 kits (divided into three experimental groups) to different quantities of prairie dog in the diet: no prairie dog, prairie dog three times per week, and prairie dog daily during the assumed sensitive period for olfactory imprinting, i.e., between 60–90 postnatal days. At age 5 months, kits were individually tested in a food choice cafeteria trial. Results indicated that higher amounts of prairie dog in the ferrets' early diet led to a higher preference for this food item when ferrets reached adulthood. These results have important implications for black-footed ferret recovery and have been considered in the reintroduction protocol. © 1996 Wiley-Liss, Inc.     

Experimentally induced plague infection in the northern grasshopper mouse (Onychomys leucogaster) acquired by consumption of infected prey

In this study, 20 laboratory reared Onychomys leucogaster from a parental population that is naturally exposed to plague were each fed a white mouse that had been inoculated with Yersinia pestis. Three of the 20 O. leucogaster died, four survived with antibody titers against Y. pestis and 13 survived with no titer against Y. pestis. In contrast, when 20 O. leucogaster from a plague naive parental population were fed infected prey, seven died and 13 survived with no antibody titer against Y. pestis. Our results suggest another means by which O. leucogaster from populations that are naturally exposed to plague may acquire the disease.     

No evidence of persistent Yersina pestis infection at prairie dog colonies in north-central Montana

Sylvatic plague is a flea-borne zoonotic disease caused by the bacterium Yersinia pestis, which can cause extensive mortality among prairie dogs (Cynomys) in western North America. It is unclear whether the plague organism persists locally among resistant host species or elsewhere following epizootics. From June to August 2002 and 2003 we collected blood and flea samples from small mammals at prairie dog colonies with a history of plague, at prairie dog colonies with no history of plague, and from off-colony sites where plague history was unknown. Blood was screened for antibody to Y. pestis by means of enzyme-linked immunosorbent assay or passive hemagglutination assay and fleas were screened for Y. pestis DNA by polymerase chain reaction. All material was negative for Y. pestis including 156 blood samples and 553 fleas from colonies with a known history of plague. This and other studies provide evidence that Y. pestis may not persist at prairie dog colonies following an epizootic.     

Serologic survey for canine infectious diseases among sympatric swift foxes (Vulpes velox) and coyotes (Canis latrans) in southeastern Colorado

Swift foxes (Vulpes velox) and coyotes (Canis latrans) are sympatric canids distributed throughout many regions of the Great Plains of North America. The prevalence of canid diseases among these two species where they occur sympatrically is presently unknown. From January 1997 to January 2001, we collected blood samples from 89 swift foxes and 122 coyotes on the US Army Pi?on Canyon Maneuver Site, Las Animas County, SE Colorado (USA). Seroprevalence of antibodies against canine parvovirus (CPV) was 71% for adult (> 9 mo old) and 38% for juvenile (< or = 9 mo old) swift foxes. Adult (<1 yr old) and juvenile (<1 yr old) coyotes had a seroprevalence for CPV of 96% and 78%, respectively. Presence of antibodies against canine distemper virus (CDV) was 5% for adult foxes and 0% for juvenile foxes. Seroprevalence of CDV was 46% for adult coyotes and 18% for juvenile coyotes. No swift foxes had canine adenovirus (CAV) antibodies, whereas 81% and 63% of adult and juvenile coyotes, respectively, had antibodies for CAV. Seroprevalence of antibodies against Yersinia pestis was 68% among adult foxes and 34% among juvenile swift foxes. Seroprevalence of Y. pestis antibodies was 90% and 70% for adult and juvenile coyotes, respectively. No swift foxes had antibodies against Francisella tularensis, whereas seroprevalence was 4% among both adult and juvenile coyotes. Antibodies against CPV and plague were common in both species, whereas antibodies against CDV and CAV were more prevalent in coyotes compared to swift foxes.     

Plague surveillance by serological testing of coyotes (Canis latrans) in Los Angeles County, California.

Sera from 19 (5.6%) of 338 coyotes (Canis latrans) collected in Los Angeles County, California between 1986 and 1990 had antibodies against Yersinia pestis using the passive hemagglutination test. Coyotes were excellent sentinels of plague activity in Los Angeles County. Enzootic plague declined dramatically in the county during the 5-yr period.     

Identification of Yersinia pestis with varied plasmid composition using monoclonal and polyclonal fluorescent immunoglobulins

Abstract The efficiency of serological identification of Yersinia pestis strains which contain different plasmids was assessed with polyclonal and monoclonal immunoglobulin preparations in the direct fluorescent antibody method. Plague polyclonal luminescent immunoglobulins recognize only those Y. pestis strains which contain pPst, pFra plasmids or both. Anticapsular plague monoclonal antibodies interact only with capsule-forming plague agent strains (pFra+) grown at 37°C. With plague monoclonal lipopolysaccharide antibodies one can identify all Y. pestis strains irrespective of their plasmid content and cultivation temperature. However, these antibodies cross-react with Yersinia pseudotuberculosis bacteria in 60% of cases. The problem of laboratory diagnosis of the plague organism, whatever its plasmid profile, can be solved through the development of a test kit involving two preparations such as plague lipopolysaccharide monoclonal luminescent antibodies and pseudotuberculosisspecific luminescent adsorbed immunoglobulins.     

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.     

Possible vector dissemination by swift foxes following a plague epizootic in black-tailed prairie dogs in northwestern Texas

To determine whether swift foxes (Vulpes velox) could facilitate transmission of Yersinia pestis to uninfected black-tailed prairie dog (Cynomys ludovicianus) colonies by acquiring infected fleas, ectoparasite and serologic samples were collected from swift foxes living adjacent to prairie dog towns during a 2004 plague epizootic in northwestern Texas, USA. A previous study (1999-2001) indicated that these swift foxes were infested almost exclusively with the flea Pulex irritans. Black-tailed prairie dogs examined from the study area harbored only Pulex simulans and Oropsylla hirsuta. Although P. irritans was most common, P. simulans and O. hirsuta were collected from six swift foxes and a single coyote (Canis latrans) following the plague epizootic. Thus, both of these canids could act as transport hosts (at least temporarily) of prairie dog fleas following the loss of their normal hosts during a plague die-off. All six adult swift foxes tested positive for antibodies to Y. pestis. All 107 fleas from swift foxes tested negative for Y. pestis by mouse inoculation. Although swift foxes could potentially carry Y. pestis to un-infected prairie dog colonies, we believe they play only a minor role in plague epidemiology, considering that they harbored just a few uninfected prairie dog fleas (P. simulans and O. hirsuta).