A coordinated recovery effort has been underway for the the Black-footed Ferret (BFF) for over thirty years. In 2019, Revive & Restore began the experimental work needed to determine whether genomic technologies can help the BFF survive outside the efforts of conservation.
As one of America’s most endangered species, and twice feared extinct, the BFF is threatened by what is called the “extinction vortex.” This is a weakening of a species’ genetic fitness brought on by progressive inbreeding, genetic drift, and an overall loss of genetic diversity. The BFF is now a model in the development of a variety of genetic rescue techniques—techniques that may also help in the recovery of other endangered species.
Saving a Species
In 2013, Revive & Restore was invited by the United States Fish & Wildlife Service’s National Black-footed Ferret Conservation Center to explore the potential use of genomic technologies to increase BFF genetic diversity. Our evaluation began in 2014 with a study of BFF genomics, and in 2016 Revive & Restore submitted a proposal to apply new techniques of genetic rescue to the problems preventing the species’ long-term sustainability: ongoing loss of genetic diversity and susceptibility to disease. In 2018, Revive & Restore received an Endangered Species Recovery permit by the USFWS to explore the potential use of genomic technologies to solve both of these problems.
The BFF has been listed as an endangered species since 1967; more than a half-century of misguided predator control campaigns and efforts to eradicate prairie dogs from farm and ranch land decimated the population. By 1981, the BFF was on the verge of extinction. But the species was saved by a bold conservation and captive breeding program initiated by USFWS (the agency responsible for overseeing the conservation of all endangered species). Conservationists rescued the last ferrets surviving in the wild and bred them in captivity to build up their numbers. BFFs were then reintroduced to their historic habitats.
Despite significant progress in re-establishing wild populations, sustaining those populations and removing the BFF from the federal endangered species list remains elusive. A five-year review of recovery efforts conducted by USFWS in November 2008 found that the species remained one of the most endangered mammals in the U.S. because of two major obstacles: Sylvatic plague and the ongoing loss of genetic diversity.
partnerships FOR THE BLACK-FOOTED FERRET
Revive & Restore partnered with San Diego Zoo Global to study the genomes of four unique BFF specimens with the goal of understanding the extent of the genetic diversity problem and whether existing genetic resources could restore diversity into the ferret population. Those four genomes were from a living ferret representative of the current population, two cryopreserved cell lines of ferrets originally captured from the wild in the 1980s that were not part of the captive breeding population, and a ferret born from an early genetic rescue attempt to recover historic diversity in which a captive-breed female was artificially inseminated using 20-year-old cryopreserved sperm. The study found that historic genetic diversity was recovered through that early genetic rescue effort and could be increased further if historic samples were bred back into the population. Cloning could be used to reproduce the historic ferrets and introduce new founding genetics into the population – a first for an endangered species.
USFWS also asked Revive & Restore to develop a long-term solution to Sylvatic Plague. While vaccines for the disease have been developed, a permanent solution is needed for ferrets to survive in the wild without human assistance. Revive & Restore then worked for the next two years with a diverse team of scientists to construct plausible solutions to Sylvatic Plague. In November 2016, Revive & Restore submitted a proposal to the National Black-footed Ferret Conservation Center outlining the initial laboratory steps needed to address the genetic rescue of the BFF on two fronts: developing genetic resistance to Sylvatic Plague and optimizing the reproductive technologies necessary to increase genetic diversity. With increased genetic variability and a decreased susceptibility to Sylvatic Plague, the recovery effort may one day achieve the goal of sustainable wild populations of BFFs across their historic range.
In 2018, Revive & Restore received a permit under the Endangered Species Act, authorizing the lab work necessary to demonstrate that the genetic rescue of the BFF is possible. Specifically, the permit allows Revive & Restore and our partners to conduct two principal activities: check the viability of using cloning techniques to bring the frozen cell lines and their genetic diversity back into the population, and test methods to convert the vaccine for plague in ferrets into a permanent inheritable trait. Importantly, Revive & Restore’s current permit from the USFWS meets the public review standards of the National Environmental Policy Act that encourages public and agency review of the proposed activities. We look forward to future rounds of public engagement on the genetic rescue of the BFF.
PROBLEMS PREVENTING FULL RECOVERY
Of the handful of wild ferrets brought into captivity between 1985 and 1987, some were members of the same family and others died before successfully breeding. It is estimated that all living Black-footed Ferrets today trace their ancestry to just seven founders. Not all of these founders contributed equally to the genetic makeup of living ferrets, despite careful efforts to retain founding diversity. Models predict that today’s ferrets possess about 85% of their original genetic diversity, and that this number will continue to fall, due to genetic drift. Loss of genetic diversity can have significant long-term consequences; it can limit adaptability to wild conditions, increase susceptibility to disease, decrease fertility, and increase the frequency of genetic abnormalities.
Even if genetic diversity is improved, the ultimate survival of wild Black-footed Ferret populations may still be jeopardized by disease. Sylvatic Plague is the single largest challenge to the species. The plague bacteria, Yersinia pestis, was brought to North America in the late 1800s, and it is the same pestilence that devastated Europe in the 14th century. Today the disease is widespread over the western United States and Great Plains.
Plague can be spread a number of ways, but ferrets contract the disease primarily from their prey, the Prairie Dog. Prairie Dog populations are large enough and diverse enough to adapt to plague, and there is evidence that some populations already exhibit resistance. This is not possible for the Black-footed Ferret. It is thought that, although the domestic ferret appears to be immune to plague, Black-footed Ferrets have always been highly susceptible. That fact, coupled with the lack of genetic diversity indicate that intervention is needed to achieve full recovery.
Plague’s complex epidemiology and widespread abundance on the landscape make it impossible to eradicate. This reality coupled with the ferret’s extreme susceptibility to the disease means successful recovery of the Black-footed Ferret is dependent upon finding a solution to that susceptibility. The consensus of dozens of scientists and conservationists is that we cannot treat any link in the chain of disease transmission, we need to treat the ferrets directly.
The good news is that a vaccine has been developed that provides lifelong immunity. That a workable vaccine has been developed means that the Black-footed Ferret’s immune system can be trained to recognize and fight plague. The vaccine can save individual ferrets, but, since vaccinated immunity cannot be inherited by wild born ferrets, it keeps the species conservation reliant. Wild-born ferrets must be captured and vaccinated, and then recaptured for a booster shot. For the species to fully recover, a genetic solution that conveys inheritable resistance is necessary.
HOW GENETIC RESCUE CAN HELP
While the BFF is susceptible to Sylvatic Plague, the domestic ferret (a close cousin) is completely resistant to the disease. We have proposed conducting a series of comparative analyses to discover which genetic traits endow domestic ferrets with immunity. Then precise genome editing would be used to write the genetic codes of those traits into the Black-footed Ferret genome. All of the laboratory research can be completed in vitro without working with living BFFs. We intend to perform disease screening in cell cultures contained in the lab. By using cell lines, animal testing will be avoided and multiple genetic solution tests can be run simultaneously at low cost.
The first step in assessing our hypotheses will be to compare the BFF’s genome with that of the domestic ferret in order to identify which genes in the immune system are different. Those genes will be likely candidates for a genomic solution. The next step will be to analyze how cellular mechanisms work when plague bacteria fail to infect domestic ferret cells and how those mechanisms change when BFF cells are infected. From these comparisons we should be able to identify a narrow set of genes involved in the immune system to fight plague. Those genes will then be edited into BFF cell lines, and disease screening assays will follow.
Once a BFF cell line exhibiting resistance to Sylvatic Plague has been produced, living ferrets will be generated by cloning for the next research step. We have proposed simultaneous experiments to optimize the process of cloning BFFs using domestic ferrets as surrogate egg cell donors and mothers, thus avoiding using endangered BFFs for experimental procedures. This application of interspecies cloning will also effectively enable the Recovery Team to increase the wild population of ferrets and conserve genetic diversity in an additive, rather than detracting, manner. It will even allow the recovery of historic genetic diversity and add new founders for the population.
Our leading hypothesis is that domestic ferrets produce Sylvatic Plague antibodies that enable an innate immune response instead of a slower adaptive immune response like the Black-footed Ferret. Introducing new antibody chains is becoming practically routine in the field of genome editing; genetic engineering of antibodies for human health purposes has steadily advanced over the past few decades using many model organisms including rodents, cattle, and even chickens. Through a similar process, we can efficiently introduce in vitro antibody elements from the domestic ferret into the immunity genes of Black-footed Ferret cells.
A second hypothesis is that cell membrane receptors are the key to the domestic ferret’s resistance to plague. Rather than fight the infection after it invades, particular changes to cell membrane receptors can completely prevent plague bacteria from infecting cells.
A third route is also possible. Plague bacteria invade immune system cells and use the host’s own cell proteins to protect themselves from being digested inside the host’s cells, a process called phagocytosis. This protection allows plague bacteria to multiply unnoticed inside the very immune system cells that are supposed to kill the bacteria. The domestic ferret’s immune cell proteins may have mutations that make them incompatible with plague’s commandeering abilities.