PROGRESS TO DATE
Catalytic Science, 2020
After permitting was received in 2018, three Catalytic Science Fund projects were begun in 2019 and continue in 2020. These three projects will establish the foundational research needed for restoring lost genetic diversity to the BFF population and for facilitating inherited immunity against sylvatic plague.
Restoring Genetic Diversity
The first project has begun analyzing will check the viability of cloning techniques via domestic ferret surrogates to bring back genetic variation from two 30-year-old cryopreserved BFF cell lines and breed it into the wild population. This genetic variability is currently held in the San Diego Zoo Global Frozen Zoo.. The success of this project would recover the genetic variation lost during the past 20-plus generations of captive breeding.
Project partners Viagen Pets and Marshall BioResources have begun cloning research using test cell lines and domestic ferret surrogate mothers. If the interspecies pregnancy viability is comparable to intraspecies results (domestic ferret embryos implanted into domestic surrogate mothers), testing of historic cell lines will proceed.
These tests are analyzing the viability of interspecies pregnancy only. If pre-parturition analyses appear promising, we will move forward with further permit applications to bring cloned embryos to term and officially revive these important individuals.
Removing Canine Distemper Virus
Of the two cell lines that are cryopreserved, one is infected with Canine Distemper Virus. This has lead to the second project: clearing these cells of viral infection so that they may also be used for cloning. ImQuest Biosciences is currently working to test methods of cleaning the virus from experimentally infected BFF cells. Once an effective method is developed, the team will move onto treating the historic cells.
Facilitating inherited immunity against sylvatic plague
Lastly, Revive & Restore has developed a research pathway to test if upregulating antibody expression for lifelong plague immunity offers a solution to establishing inheritable immunity to the disease and eliminating the need for vaccination. The plan is to test this method in mice as a first step. Texas A&M Institute for Genomic Medicine are developing these mice for plague challenges at the USGS National Wildlife Health Center. This first-of-its-kind study may also lead to a means for establishing innate, inheritable immunity to other exotic bacterial diseases in diverse mammal species.
Endangered Species Recovery Permit, 2018
In 2018, Revive & Restore received a first-of-its-kind Endangered Species Recovery Permit from the United States Fish & Wildlife Service to initiate the genetic rescue of the black-footed ferret. The permit authorizes the lab work necessary to demonstrate that the genetic rescue of black-footed ferret is possible. Specifically, the permit allows Revive & Restore and our partners to conduct two principal activities. First, we will check the viability of using cloning techniques to bring 30-year-old frozen cell lines, currently stored in the San Diego Zoo Global Frozen Zoo, and their genetic diversity back into the wild population. Revive & Restore and our partners will also test methods to convert the vaccine for plague in ferrets into a permanent heritable 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 black-footed ferret.
Earlier Genomic Study
Genomics can be used in many of ways to improve the conservation of the BFF.
BFF breeding has been intensely managed using theoretical measures of genetic diversity based on kinship. But because all living ferrets share the same ancestry at a kinship level between first cousins and siblings, it very difficult to choose mate pairs. Genome sequencing can create an empirical decision matrix that will allow breeders to selecting mate-pairs based on the unique genetic contribution their offspring will receive, rather than on kinship. Whole genome analyses can identify maladaptive genes responsible for traits associated with inbreeding depression, such as low sperm mobility, cryptorchidism, and kinked tails, and prevent the accumulation of these traits in the population. Studying genomes of wild-born ferrets can help identify what alleles are selected in the wild in order to breed ferrets in captivity more suited to their particular release sites.
Maximizing the genomic technology benefits in the recovery of BFFs requires building a databank of genomic knowledge. Revive & Restore initiated this step in 2014 in partnership with San Diego Zoo Global’s (SDZG) Frozen Zoo and Cofactor Genomics. Revive & Restore sequenced the genomes of four, carefully selected BFF samples to help answer these preliminary questions:
- How genetically diverse was the last wild population of BFFs that founded the captive breeding program?
- Has overall genomic diversity of the BFF decreased over the past 30 years?
- Would integrating historic BFF individuals restore genomic diversity?
- How much diversity might be gained with continued genetic rescue efforts?
The study individuals include two unique individuals captured at Meeteetse, Wyoming in the 1980s, a living ferret born in 2009 whose genetics is representative of the current captive population, and a unique individual born in 2010 as the result of artificial insemination using semen cryopreserved in the 1980s. (This was an early genetic rescue attempt to restore lost diversity.) The DNA samples of two living ferrets born in captivity were provided by the BFF Recovery Team, while the Frozen Zoo provided the historic samples for the two individuals born in the wild. Both of these ferrets were members of the population that founded the captive breeding program, so their genomes represent a sample of the genetic diversity of the founding generation of all living ferrets. However, these individual ferrets’ unique genetic lineages were lost to the captive breeding program because they did not produce viable offspring before they died. Their cryopreserved cells are a potential resource for restoring lost genetic diversity, and for infusing new genetic variation through cloning.
2014 - 2016
The Study Individuals
Studbook number SB6573 was a male named Cheerio, born 2009. Cheerio’s genome shares ancestry from all seven founders, presumably in equal amounts. All living ferrets are presumed to share similar ancestry to all seven founders, thus Cheerio is the proverbial “every ferret” of the living population for this research. Cheerio died shortly after our genomic study, after a long and productive life siring 10 kits to continue Black-footed Ferret recovery.
Studbook number SB6815 was a male named Balboa, born 2010. Balboa is unique; he was born by artificial insemination, using the cryopreserved sperm of Rocky, a ferret captured in the 1980s at Meeteetse and assumed to be the son of one of the founders, Scarface. This form of “cryogenic artificial insemination” is a viable genetic rescue method, partially restoring the genetic diversity lost over 20 years and 11 generations. Balboa died in spring 2016. He sired 4 kits through natural breeding, which are the 4 most genetically diverse living Black-footed Ferrets.
Studbook Number 2 was an unnamed wild male captured at Meeteetse in 1985. This male was among the first six wild ferrets captured for captive breeding, but all six died of canine distemper before they could be bred in captivity. However, tissue samples of this male were shipped to the San Diego Frozen Zoo where cell cultures were grown and cultured.
Studbook number SB10 was a wild female named Willa, captured at Meeteetse between 1986 and 1987. Willa successfully bred in captivity, but her kits died without leaving any descendants.
For each of the four BFF study individuals, 90 percent, or 2.2 billion base pairs of DNA, were mapped successfully to the publicly available domestic ferret reference genome. The genomic analysis found that the level of diversity between the four individuals amounted to approximately 2 million variable mutations, which is roughly similar to overall diversity exhibited by other endangered carnivores. Some of these variations were shared between two or three of the study individuals, but some were completely unique to specific individuals. By comparing the shared and unique diversity of the four ferrets, we found that BFFs in the 1980s possessed more genetic diversity than living ferrets, suggesting that some diversity has been lost by the breeding pedigree, as predicted. A secondary analysis further confirmed that living ferrets have suffered a small degree of inbreeding.
However, the results also showed that genomic diversity can be improved through techniques of genetic rescue. The genome of Balboa, the study individual born via artificial insemination and advanced reproductive techniques, was as diverse as that of Willa, a ferret born in the wild and captured for the captive breeding program in the 1980s. This indicates that it is possible to slow, or possibly reverse, the genetic erosion of the captive breeding program. Cloning the two cell cultures housed in the Frozen Zoo and integrating them into to the breeding program would potentially introduce a substantial amount of unique diversity relative to the diversity of living ferrets.
With only four individuals analyzed, it is not yet possible to know how much variation has truly been lost from the seven captive breeding founders and how much of that variation can be restored through cloning or artificial insemination. In order to answer these questions and to begin researching genetic rescue techniques for Sylvatic plague resistance, Revive & Restore and SDZG has partnered with the lab of Federica DiPalma at the Earlham Institute to compare more individual BFFs. The DiPalma group has sequenced the genomes of four individuals born in the wild that descended from successful reintroductions at Shirley Basin, Wyoming and Badlands National Park, South Dakota.
Thus far, the DiPalma group has sequenced DNA from over 90 strains of domestic ferret as well as other wild species of ferret, including the Siberian Polecat, the closest relative to the Black-footed Ferret, which is also susceptible to Sylvatic Plague. With a focus on the genetics of the immune system (termed the immunome), the DiPalma group has improved our understanding of the genes that comprise ferret immune systems and of their evolution. By comparing the immunomes of a diverse sample of individuals of these three ferret species, we can begin to identify the genes that may be involved in Sylvatic Plague resistance and susceptibility. From our preliminary study, we’ve found that the Black-footed Ferret and domestic ferret genomes are approximately 99.6% identical, meaning the key differences that make domestic ferrets resistant to Sylvatic Plague is contained in a small number of mutations.