Markers of fertility in reproductive microbiomes of male and female endangered black-footed ferrets (Mustela nigripes).

Reproductive microbiomes contribute to reproductive health and success in humans. Yet data on reproductive microbiomes, and links to fertility, are absent for most animal species. Characterizing these links is pertinent to endangered species, such as black-footed ferrets (Mustela nigripes), whose populations show reproductive dysfunction and rely on ex-situ conservation husbandry. To understand microbial contributions to animal reproductive success, we used 16S rRNA amplicon sequencing to characterize male (prepuce) and female (vaginal) microbiomes of 59 black-footed ferrets at two ex-situ facilities and in the wild. We analyzed variation in microbiome structure according to markers of fertility such as numbers of viable and non-viable offspring (females) and sperm concentration (males). Ferret vaginal microbiomes showed lower inter-individual variation compared to prepuce microbiomes. In both sexes, wild ferrets harbored potential soil bacteria, perhaps reflecting their fossorial behavior and exposure to natural soil microbiomes. Vaginal microbiomes of ex-situ females that produced non-viable litters had greater phylogenetic diversity and distinct composition compared to other females. In males, sperm concentration correlated with varying abundances of bacterial taxa (e.g., Lactobacillus), mirroring results in humans and highlighting intriguing dynamics. Characterizing reproductive microbiomes across host species is foundational for understanding microbial biomarkers of reproductive success and for augmenting conservation husbandry.

Gut bacterial composition shows sex-specific shifts during breeding season in ex situ managed black-footed ferrets.

The gut microbiome of mammals engages in a dynamic relationship with the body and contributes to numerous physiological processes integral to overall health. Understanding the factors shaping animal-associated bacterial communities is therefore paramount to the maintenance and management in ex situ wildlife populations. Here, we characterized the gut microbiome of 48 endangered black-footed ferrets (Mustela nigripes) housed at Smithsonian's National Zoo and Conservation Biology Institute (Front Royal, Virginia, USA). We collected longitudinal fecal samples from males and females across two distinct reproductive seasons to consider the role of host sex and reproductive physiology in shaping bacterial communities, as measured using 16S rRNA amplicon sequencing. Within each sex, gut microbial composition differed between breeding and non-breeding seasons, with five bacterial taxa emerging as differentially abundant. Between sexes, female and male microbiomes were similar during non-breeding season but significantly different during breeding season, which may result from sex-specific physiological changes associated with breeding. Finally, we found low overall diversity consistent with other mammalian carnivores alongside high relative abundances of potentially pathogenic microbes such as Clostridium, Escherichia, Paeniclostridium, and (to a lesser degree) Enterococcus - all of which have been associated with gastrointestinal or reproductive distress in mammalian hosts, including black-footed ferrets. We recommend further study of these microbes and possible therapeutic interventions to promote more balanced microbial communities. These results have important implications for ex situ management practices that can improve the gut microbial health and long-term viability of black-footed ferrets.

EVALUATING BAITS WITH LUFENURON AND NITENPYRAM FOR FLEA CONTROL ON PRAIRIE DOGS (CYNOMYS SPP.) TO MITIGATE PLAGUE.

Plague, caused by Yersinia pestis, is a widespread threat to endangered black-footed ferrets (Mustela nigripes) and their primary prey, prairie dogs (Cynomys spp.). Wildlife biologists most commonly manage plague using insecticides to control fleas, the primary vectors of Y. pestis. We tested edible baits containing the insecticides lufenuron and/or nitenpyram in prairie dogs. During a laboratory study, we treated 26 white-tailed prairie dogs (Cynomys leucurus) with lufenuron at 300 mg/kg body mass. All animals remained clinically healthy over the 9 wk monitoring period. Although serum lufenuron concentrations were >130 ppb in two treatment groups at week 1, concentrations declined to ≤60 ppb after 3 wk in non-torpid prairie dogs and after 7 wk in torpid prairie dogs. In a field experiment, we tested baits containing a combination of 75 mg lufenuron and 6 mg nitenpyram, respectively, in black-tailed prairie dogs (Cynomys ludovicianus). We uniformly distributed baits at 125 baits/ha on two plots (treated once) and 250 baits/ha on two plots (each treated twice 4.4 wk apart). Following treatments, flea abundance increased on prairie dogs and remained stable in burrows. Our findings indicate that baits containing lufenuron and nitenpyram, at the reported treatment rates, are ineffective tools for flea control on prairie dogs. Future experiments might evaluate efficacy of higher doses of lufenuron and nitenpyram, and repetitive treatments at differing intervals over time to evaluate potentially therapeutic treatments.

Plague mitigation for prairie dog and black-footed ferret conservation: Degree and duration of flea control with 0.005% fipronil grain bait.

Sylvatic plague, a primarily flea-borne zoonosis, is a significant threat to prairie dogs (Cynomys spp., PDs) and their specialized predators, endangered black-footed ferrets (Mustela nigripes, BFFs). Host-fed fipronil baits have proven effective in controlling fleas on PDs for the purposes of plague mitigation and BFF conservation. Currently, annual treatments are the norm. We tested the long-term efficacy of fipronil bait treatments with black-tailed PDs (C. ludovicianus, BTPDs) and BFFs in South Dakota, USA. During 2018-2020, we provided BTPDs on 21 sites with grain bait formula, laced with 0.005% fipronil (50 â€‹mg/kg); 18 non-treated sites functioned as baselines. In 2020-2022, we live-trapped, anesthetized, and combed BTPDs for fleas. Flea control was significant for at least 639-885 days. Flea abundance on the treated sites was < 0.5 fleas/BTPD for ∼750 days. During 2020-2022, we sampled BFFs for fleas on 4 BTPD colonies treated with fipronil grain bait and 8 non-treated colonies. Flea control was significant with BFFs, but flea abundance began to rebound within ∼240 days post-treatment. When feasible, the combination of insecticide treatments, such as fipronil baits, and BFF vaccination against plague provide a "two-pronged" protection approach for these endangered carnivores. If fipronil bait treatments are less effective with predatory BFFs than PDs, as found herein, the "two-pronged" approach might be used to protect BFFs and biennial fipronil bait treatments might be used to protect PDs. If BFF vaccination is not possible, or few BFFs can be vaccinated, annual fipronil bait treatments might be used as a precaution to protect BFFs. Flea densities might be surveyed to determine when/where more frequent treatments seem useful.

FLEA CONTROL ON PRAIRIE DOGS (CYNOMYS SPP.) WITH FIPRONIL BAIT PELLETS: POTENTIAL PLAGUE MITIGATION TOOL FOR RAPID FIELD APPLICATION AND WILDLIFE CONSERVATION.

Sylvatic plague is a widespread, primarily flea-vectored disease in western North America. Because plague is highly lethal to endangered black-footed ferrets (Mustela nigripes, BFFs) and the prairie dogs (Cynomys spp., PDs) on which BFFs depend for habitat and prey, minimizing the impacts of plague is a priority at BFF reintroduction sites. We developed a new, flour-based bait pellet containing 0.84 mg of fipronil and weighing ∼1.25 g (FipBits). We measured the degree and duration of flea control on black-tailed PDs (C. ludovicianus) in Montana and on Gunnison's PDs (C. gunnisoni) in Arizona, USA from 2018-2020. FipBits were distributed on treated plots one time at a rate of 125/ha. Fleas were virtually eliminated in Montana from 1 mo posttreatment to 1 yr later and remained substantially depressed 2 yr posttreatment. With the split colony design, we probably underestimated the degree of flea control achieved with FipBits due to crossover edge effects along the arbitrary line dividing the plots. Flea control in Arizona was significant from 1 mo posttreatment to 1 yr later, but flea abundance had recovered by 2 yr posttreatment. Flea control was evaluated from 2020-2021 in South Dakota, USA on four plots treated with three concentrations of fipronil in FipBits (0.68, 0.71, and 0.83 mg/FipBit). Fleas were essentially eliminated for 10 mo on the 0.83-mg plot and were substantially reduced on the two 0.71-mg plots. Fleas were reduced on the 0.68-mg plot, but the degree of control was less than observed on other treated plots. Impacts of plague on PDs and BFFs would probably be greatly reduced by the levels of flea control observed with FipBits. Options for expanded FipBit evaluations are being pursued for what may become a highly practical, affordable, and effective plague mitigation tool.

Assembling a safe and effective toolbox for integrated flea control and plague mitigation: Fipronil experiments with prairie dogs.

BACKGROUND: Plague, a widely distributed zoonotic disease of mammalian hosts and flea vectors, poses a significant risk to ecosystems throughout much of Earth. Conservation biologists use insecticides for flea control and plague mitigation. Here, we evaluate the use of an insecticide grain bait, laced with 0.005% fipronil (FIP) by weight, with black-tailed prairie dogs (BTPDs, Cynomys ludovicianus). We consider safety measures, flea control, BTPD body condition, BTPD survival, efficacy of plague mitigation, and the speed of FIP grain application vs. infusing BTPD burrows with insecticide dusts. We also explore conservation implications for endangered black-footed ferrets (Mustela nigripes), which are specialized predators of Cynomys. PRINCIPAL FINDINGS: During 5- and 10-day laboratory trials in Colorado, USA, 2016-2017, FIP grain had no detectable acute toxic effect on 20 BTPDs that readily consumed the grain. During field experiments in South Dakota, USA, 2016-2020, FIP grain suppressed fleas on BTPDs for at least 12 months and up to 24 months in many cases; short-term flea control on a few sites was poor for unknown reasons. In an area of South Dakota where plague circulation appeared low or absent, FIP grain had no detectable effect, positive or negative, on BTPD survival. Experimental results suggest FIP grain may have improved BTPD body condition (mass:foot) and reproduction (juveniles:adults). During a 2019 plague epizootic in Colorado, BTPDs on 238 ha habitat were protected by FIP grain, whereas BTPDs were nearly eliminated on non-treated habitat. Applications of FIP grain were 2-4 times faster than dusting BTPD burrows. SIGNIFICANCE: Deltamethrin dust is the most commonly used insecticide for plague mitigation on Cynomys colonies. Fleas on BTPD colonies exhibit the ability to evolve resistance to deltamethrin after repeated annual treatments. Thus, more tools are needed. Accumulating data show orally-delivered FIP is safe and usually effective for flea control with BTPDs, though potential acute toxic effects cannot be ruled out. With continued study and refinement, FIP might be used in rotation with, or even replace deltamethrin, and serve an important role in Cynomys and black-footed ferret conservation. More broadly, our stepwise approach to research on FIP may function as a template or guide for evaluations of insecticides in the context of wildlife conservation.

RECOMBITEK CANINE DISTEMPER VACCINE AS AN ALTERNATIVE FOR PUREVAX DISTEMPER VACCINE IN ENDANGERED BLACK-FOOTED FERRETS (MUSTELA NIGRIPES).

Black-footed ferrets (Mustela nigripes) are an endangered species in North America that are highly sensitive to canine distemper virus (CDV) infections and any exposure could be devastating to species recovery. The U.S. Fish and Wildlife Service recovery program has safely used a recombinant DNA (rDNA) canarypox-vectored CDV vaccine, Purevax® Ferret Distemper (PFD), to vaccinate black-footed ferrets. Because of a PFD shortage in 2015, an rDNA vaccine labeled for use in dogs, Recombitek® CDV (rCDV), was chosen to vaccinate black-footed ferrets. Our goal was to compare the serum neutralizing (SN) titers after vaccination of 17 captive and 18 wild black-footed ferrets with rCDV or PFD, respectively, considering ≥1:128 as a protective titer. Both vaccines produced comparable 1 yr postvaccination protective titers in captive and wild black-footed ferrets. In wild black-footed ferrets, one PFD vaccination produced SN titers similar to two PFD vaccinations at 1 yr postvaccination. One year after vaccination with rCDV, SN titers in captive black-footed ferrets were higher than in wild ferrets. These results indicate rCDV may be an effective alternative CDV vaccine in captive black-footed ferrets and PFD should be prioritized for wild ferrets because one dose was effective for animals that can be difficult to recapture.

Fipronil Pellets Reduce Flea Abundance on Black-Tailed Prairie Dogs: Potential Tool for Plague Management and Black-Footed Ferret Conservation.

In western North America, sylvatic plague (a flea-borne disease) poses a significant risk to endangered black-footed ferrets (Mustela nigripes) and their primary prey, prairie dogs (Cynomys spp.). Pulicides (flea-killing agents) can be used to suppress fleas and thereby manage plague. In South Dakota, US, we tested edible "FipBit" pellets, each containing 0.84 mg fipronil, on free-living black-tailed prairie dogs (Cynomys ludivicianus). FipBits were applied along transects at 125 per ha and nearly eliminated fleas for 2 mo. From 9-14 mo post-treatment, we found only 10 fleas on FipBit sites versus 1,266 fleas on nontreated sites. This degree and duration of flea control should suppress plague transmission. FipBits are effective, inexpensive, and easily distributed but require federal approval for operational use.

Managing plague on prairie dog colonies: insecticides as ectoparasiticides.

Human health practitioners and wildlife biologists use insecticides to manage plague by suppressing fleas (Siphonaptera), but insecticides can also kill other ectoparasites. We investigated effects of deltamethrin and fipronil on ectoparasites from black-tailed prairie dogs (Cynomys ludovicianus, BTPDs). In late July, 2018, we treated three sites with 0.05% deltamethrin dust and 5 sites with host-fed 0.005% fipronil grain. Three non-treated sites functioned as experimental baselines. We collected ectoparasites before treatments (June-July, 2018) and after treatments (August-October, 2018, June-July, 2019). Both deltamethrin and fipronil suppressed fleas for at least 12 months. Deltamethrin had no detectable effect on mites (Arachnida). Fipronil suppressed mites for at least 12 months. Lice (Phthiraptera) were scarce on non-treated sites throughout the study, complicating interpretation. Concentrating on eight sites where all three ectoparasites where found in June-July, 2018 (before treatments), flea intensity was greatest on BTPDs carrying many lice and mites. These three ectoparasites co-occurred at high numbers, which might facilitate plague transmission in some cases. Lethal effects of insecticides on ectoparasite communities are potentially advantageous in the context of plague management.

Evaluation of Five Pulicides to Suppress Fleas on Black-Tailed Prairie Dogs: Encouraging Long-Term Results with Systemic 0.005% Fipronil.

Plague, a flea-borne disease, hampers efforts to restore populations of black-footed ferrets (Mustela nigripes), which occupy colonies of prairie dogs (Cynomys spp.) in North America. Plague is managed by infusing prairie dog burrows with DeltaDust® 0.05% deltamethrin, a pulicide that kills fleas. Experiments are needed to identify pulicides that can be used in rotation with DeltaDust for integrated plague management. In South Dakota, USA, we tested the efficacy of four pulicide dusts when applied at a rate of 8 g per burrow on colonies of black-tailed prairie dogs (Cynomys ludovicianus): Sevin® 5% carbaryl; Dusta-cide® 6% malathion; Alpine® 0.25% dinotefuran with 95% diatomaceous earth; and Tri-Die® 1% pyrethrum with 40% amorphous silica and 10% piperonyl butoxide. We also tested systemic 0.005% fipronil, which was distributed as ½ cup of laced grain per burrow. We sampled prairie dogs on 3294 occasions and detected 10,041 fleas. Sevin and Dusta-cide suppressed fleas but only for 1 month. Neither Alpine nor Tri-Die had any noticeable, consistent effect on fleas. Fipronil suppressed fleas by 97-100% for 3 months. The residual effect of fipronil persisted for ∼12 months. Efficacy of fipronil seems comparable with DeltaDust, which exhibited a residual effect for ∼10 months in prior studies. Continued research is needed to optimize fipronil treatments for plague management on prairie dog colonies.

RESISTANCE TO DELTAMETHRIN IN PRAIRIE DOG ( CYNOMYS LUDOVICIANUS) FLEAS IN THE FIELD AND IN THE LABORATORY.

Sylvatic plague poses a substantial risk to black-tailed prairie dogs ( Cynomys ludovicianus) and their obligate predator, the black-footed ferret ( Mustela nigripes). The effects of plague on prairie dogs and ferrets are mitigated using a deltamethrin pulicide dust that reduces the spread of plague by killing fleas, the vector for the plague bacterium. In portions of Conata Basin, Buffalo Gap National Grassland, and Badlands National Park, South Dakota, US, 0.05% deltamethrin has been infused into prairie dog burrows on an annual basis since 2005. We aimed to determine if fleas ( Oropsylla hirsuta) in portions of the Conata Basin and Badlands National Park have evolved resistance to deltamethrin. We assessed flea prevalence, obtained by combing prairie dogs for fleas, as an indirect measure of resistance. Dusting was ineffective in two colonies treated with deltamethrin for >8 yr; flea prevalence rebounded within 1 mo of dusting. We used a bioassay that exposed fleas to deltamethrin to directly evaluate resistance. Fleas from colonies with >8 yr of exposure to deltamethrin exhibited survival rates that were 15% to 83% higher than fleas from sites that had never been dusted. All fleas were paralyzed or dead after 55 min. After removal from deltamethrin, 30% of fleas from the dusted colonies recovered, compared with 1% of fleas from the not-dusted sites. Thus, deltamethrin paralyzed fleas from colonies with long-term exposure to deltamethrin, but a substantial number of those fleas was resistant and recovered. Flea collections from live-trapped prairie dogs in Thunder Basin National Grassland, Wyoming, US, suggest that, in some cases, fleas might begin to develop a moderate level of resistance to deltamethrin after 5-6 yr of annual treatments. Restoration of black-footed ferrets and prairie dogs will rely on an adaptive, integrative approach to plague management, for instance involving the use of vaccines and rotating applications of insecticidal products with different active ingredients.

The black-footed ferret: on the brink of recovery?

In an attempt to save the species from extinction, the last remaining 18 black-footed ferrets (Mustela nigripes) were trapped up from the wild to initiate a captive breeding program. Nearly 30 years later more than 8,000 black-footed ferrets have been produced in captivity and approximately 4,100 animals have been reintroduced into 20 sites in eight US states (Arizona, New Mexico, Utah, Colorado, Kansas, Wyoming, South Dakota and Montana), Mexico and Canada. However, full recovery of the species has yet to be achieved, mainly due to limited viable habitat, disease and reduced fecundity. This chapter will highlight the advances in the black-footed ferret recovery program over the last 10 years including: (1) adaptive management techniques employed for the captive population; (2) development of new reintroduction sites and associated challenges facing wild black-footed ferrets; and (3) optimization of assisted reproductive techniques to secure the future of this rare species.

Ectoparasites in black-footed ferrets (Mustela nigripes) from the largest reintroduced population of the Conata Basin, South Dakota, USA.

The black-footed ferret, Mustela nigripes, is an endangered carnivore endemic to the grasslands of North America. We present the first investigation of ectoparasites associated with black-footed ferrets since reintroduction. We sampled more than 200 individuals from one of the largest and most successful reintroduced populations located in the Conata Basin of South Dakota, USA. We compared our findings with ectoparasite assemblages of sympatric carnivores and historic ferret records. We collected more than 1,000 ectoparasites consisting mainly of three flea and tick species, two of which were known historically from South Dakota. Despite our extensive sampling efforts, we did not detect any lice. This is notable because a putative host-specific louse, Neotrichodectes sp., was presumed to have gone extinct when black-footed ferrets were extirpated from the wild. The ectoparasite assemblage on black-footed ferrets comprised only generalist parasites, particularly those found on their prey such as prairie dogs (Cynomys sp.). Oropsylla hirsuta was the most abundant ectoparasite, representing 57% of all ectoparasites detected; a flea vector important in the persistence and transmission of plague. Black-footed ferrets like other endangered species undergo repeated parasite removal and vaccination efforts to facilitate population recovery, which may have unintentionally contributed to their depauperate ectoparasite community.

Environmental enrichment affects adrenocortical stress responses in the endangered black-footed ferret.

Potential stressors of wildlife living in captivity, such as artificial living conditions and frequent human contact, may lead to a higher occurrence of disease and reduced reproductive function. One successful method used by wildlife managers to improve general well-being is the provision of environmental enrichment, which is the practice of providing animals under managed care with environmental stimuli. The black-footed ferret (Mustela nigripes) is a highly-endangered carnivore species that was rescued from extinction by removal of the last remaining individuals from the wild to begin an ex situ breeding program. Our goal was to examine the effect of environmental enrichment on adrenocortical activity in ferrets by monitoring fecal glucocorticoid metabolites (FGM). Results demonstrated that enrichment lowered FGM in juvenile male ferrets, while increasing it in adult females; enrichment had no effect on FGM in juvenile females and adult males. These results correspond with our findings that juvenile males interacted more with the enrichment items than did adult females. However, we did not detect an impact of FGM on the incidence of disease or on the ability of ferrets to become reproductive during the following breeding season. We conclude that an environmental enrichment program could benefit captive juvenile male ferrets by reducing adrenocortical activity.