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Slideshow: What's Killing the Pygmy Rabbit

Slideshow: A Day in the Life of a Zoo Pathologist

The pygmy rabbits at Northwest Trek Wildlife Park were dying. Rapidly. On one visit to their pens at the wildlife preserve, about 95 km south of Seattle, they would be munching contentedly on fresh greens or sagebrush. On the next routine check, the tiny 400-gram rabbits, roughly the size of baby cottontails, would be dead. In some cases, the rabbits crawled into their burrows and never came out; in others, wildlife staff found them sitting, motionless.

Something killed Titon on February 6, then Minnie on February 7. That wasn't unusual for the highly endangered species, which has a mortality rate that is likely greater than 50% per year. But then the pace quickened. Wazzu died on February 22, followed by a death nearly every day, sometimes two, with little or no warning....

Gross necropsies didn't help determine the cause. Case found signs of enteritis that she thought could be viral, but also granulomas characteristic of Mycobacterium avium. The epidemiology also yielded no clues; the first three cases were in adjoining pens, but the others were scattered.

Case, who had joined Northwest Trek just a year earlier in January 2006, immediately stepped up biosecurity. "I put those keepers through hell," she says. The facility went through boxes and boxes of gloves to keep from passing infectious agents from one rabbit to another, and everyone used a separate bag to handle each animal during exams and treatment. As she made rounds, however, she realized how much stress the team was creating by repeatedly cornering a particular rabbit in a large pen. In order to keep a close eye on each animal while minimizing the stress of captures, she put them in individual kennels so they'd be easier to catch, and she converted a small shed into a makeshift hospital.

"Only occasionally did I have 12 hours, where the keeper said, 'I'm seeing soft stools,'" suggesting the animal was ill.
-Allison Case

On February 25, she began treating all of the rabbits with enterofloxacin, an antibiotic that fights a large spectrum of enteric bacteria, "knowing full well I could be dealing with a virus" that wouldn't respond to antibiotics. But the rabbits continued dying. After a brief break, the 10th and 11th rabbits died on March 15. Whatever was killing the rabbits had just done in more than half of the Northwest Trek population, which went from 20 to nine in little more than a month.

The presumed outbreak couldn't have come at a worse time. The Washington State Department of Fish and Wildlife and the US Fish and Wildlife Service were planning to release 28 rabbits in the Sagebrush Flat Wildlife Area, about 300 kilometers east of Northwest Trek. The highly anticipated release was a pilot, the first since Northwest Trek and other area preserves began breeding programs, designed to determine how rabbits raised in captivity would do in the wild.

Case called off the release of the Northwest Trek rabbits. The first-ever reintroduction of Columbia Basin pygmy rabbits into the wild included no animals living at Northwest Trek, and the number released fell from 28 to 20 rabbits, bred at programs at Washington State University (WSU) in Pullman, and the Oregon Zoo in Portland. For now, all the years of hard work by the Northwest Trek staff was for naught.

The pygmy rabbit (Brachylagus idahoensis) is unique enough to have its own genus. They're the only nondomesticated North American rabbits that burrow, and they subsist almost entirely on sagebrush, a highly pungent plant full of terpenes that are normally poisonous to most other animals. On average, adults weigh just 400 grams and are only 25 centimeters long - about the length of a pencil. Their native habitat is the Great Basin of the United States, extending from the Great Salt Lake in Utah and northwest to the state of Washington.

Pygmy rabbit behavior, says Northwest Trek education curator Dan Belting, follows a simple credo: "Breed hard, die young." They breed 2-3 times per year with an average litter size of four, and probably live as long as four years in the wild, although no one's quite sure. At least half of them die in the first year of life, picked off by predators such as weasels, coyote, badgers, bobcats, and raptors, as well as diseases such as sylvatic plague and tularemia.

WHAT'S UP, DOC? Lisa Shipley of Washington State University holds French Fry, a member of the Idaho subpopulation. "Frenchie" is one of about 80 in captivity in three breeding programs, of which WSU is one.

The reliance on sagebrush growing in deep soil - for burrowing purposes - has probably created the biggest challenge for the species in the wild. The Columbia River, which forms much of the border between Oregon and Washington, is banked throughout most of its path by rolling hills covered in sagebrush, in soil that's perfect for pygmy rabbits and terrible for farmers. As engineers dammed the Columbia River in the last century, however, increasingly more of that land became arable, squeezing the rabbits out of their habitat. Sagebrush-rich habitat that still exists is often isolated from other habitats.

Researchers who study the rabbits say that such isolation might have caused a poor genetic pool in the current Columbia Basin subpopulation, which now has just one or two alleles in many loci, according to Lisa Shipley, who runs the pygmy rabbit captive breeding facility at WSU. The theory is that the isolation caused the population to became genetically bottlenecked, and the loss of habitat magnified that effect.

The result is a population with a baseline immunocompromised status. When researchers tested rabbits who died of an outbreak at WSU of what turned out to be M. avium between June 2002 and September 2004, they found that Columbia Basin rabbits scored consistently worse than Idaho pygmy and cottontail rabbits on seven different markers of immune status, including complement CH50, interleukins, and tumor necrosis factor.

A report of that outbreak became a 2006 paper in the Journal of Zoo and Wildlife Medicine, and although the evidence for genetic bottlenecking is strong, scientists still want to confirm it. "We'd love to write a paper correlating genetic diversity with hardiness, but our geneticist was consumed by salmon 24 hours a day," says Lisa Harrenstien, an Oregon Zoo veterinarian, referring to a massive Oregon state salmon sequencing project that continues to divert the geneticist's attention.

Whatever the cause of the Columbia basin pygmies' low genetic diversity, by 1995 estimates were that just 150 wild Columbia Basin rabbits remained; by 2001, that estimate fell to 30. That year, the US Fish and Wildlife Service listed the Columbia Basin pygmy rabbit as endangered, and state and federal agencies stepped up efforts to save the species. "For some species, we don't know why they're declining; we don't know if we can do anything for them," says Dave Hays, an endangered species specialist with the Washington Department of Fish and Wildlife, who heads up the state's work to save pygmy rabbits. "Those are very frustrating. With the pygmy rabbit, we have an idea of what we can do to save them, and we wanted to take that opportunity to see if we could turn it around."

That year and the next, veterinarians captured as many Columbia Basin rabbits in Washington as they could find, and brought them to WSU and the Oregon Zoo. Their efforts yielded only 16 wild rabbits, however, and no one has reported spotting a single wild Columbia Basin pygmy rabbit anywhere for five years.

The experts got to work. Northwest Trek began building their breeding facility in 2001, after deputy director Dave Ellis heard about the Oregon Zoo's project. The US Fish and Wildlife Service and Washington Department of Fish and Wildlife, which run the reintroduction program at all three facilities, have spent close to $3 million on pygmy rabbits in the past six years, with about half for habitat acquisiton for a number of species including the pygmy rabbit.

Given the Columbia Basin rabbits' limited genetic pool, the goal of the breeding program is to cross the rabbits with a hardier stock from Idaho, resulting in a population of rabbits that are each at least genetically 75% Columbia Basin, but can survive in the wild. Vets choose hardy animals, then cross an Idaho with a Columbia Basin rabbit, resulting in 50% Columbia Basin offspring. Those are then crossed with a Columbia Basin rabbit, to generate 75% Columbia Basin offspring. Organizers were planning to release rabbits starting a few years ago, but the captive population hasn't grown quickly enough, and the March release was the first.

THE BUNNY KEEPER: Becky Elias stands by three "carports" used as large pygmy rabbit pens, to give the rabbits a sense of living in larger spaces than their usual pens.

Despite the slow results, the new rabbits show good signs of fortitude. Margot Monti of the Oregon Zoo notes that, because of inbreeding, the native Columbia Basin rabbits were often missing metacarpals and metatarsals - poor traits in burrowing animals. Crossing them with Idaho rabbits has eliminated that problem.

"Taxonomic purists would say we've given up on purebred Columbia Basin rabbits," says Mitch Finnegan, a veterinarian at the Oregon Zoo. Indeed, just one male purebred Columbia Basin rabbit (named Lolo) and two females (Raphella and Brynn) are all that remain in captivity. Lolo and Raphella are too closely related to breed, and Lolo and Brynn have been paired together a number of times unsuccessfully. If Lolo and Brynn never breed, they and Raphella will be the last purebred Columbian Basin pygmy rabbits.

It's a Wednesday afternoon in late April, and I'm driving south on a highway outside of Portland, Ore. I'm following a Chevrolet Cavalier with a "Recycle" license plate holder and a Bugs Bunny sticker on its rear windshield. Sitting in the back seat of the Chevrolet in a pet carrier, surrounded by large bags of greens and sagebrush, is a 75% Columbia Basin pygmy rabbit named Onyx. He, the car's driver, Rachel Lamson, and I are headed from the Oregon Zoo to a California condor breeding facility in Clackamas County, Ore.

THE CULPRIT? A coronavirus particle found in the stool of one of the affected Northwest Trek pygmy rabbits.

Coronavirus: courtesy of Washington State University

Onyx is moving in temporarily to the condor facility to be the subject of an experiment. He'll be released into a 185 square meter pen to see how he does in a "prerelease setting," with the hope that he might eventually be released in Sagebrush Flats and breed with the females already released. Lamson and others have already carefully constructed his pen - as they did those of other rabbits - to keep out predators. The condor pens, also carefully enclosed, are far away on the other side of the facility. "The idea is to get him used to large spaces and foraging," Lamson says, "and wean him off food pellets."

Lamson - who blushes as she tells me her colleagues sometimes call her "The Rabbit Whisperer" - spends five days a week tending to the pygmy rabbits at the zoo, and finds a reason to visit them most weekend days, even while she holds down another job working as a vet technician for a bird keeper. "I don't have much of a life," she confesses sheepishly when she opens the door of her car, which is often filled, as it is today, with rabbit food and a thin layer of dust, the remnants of the special dirt she hauls around for the rabbits.

She's been working with pygmy rabbits at the zoo since 2001. "If you show me pictures of rabbits, I can tell who they are," she says. Behavior also provides clues. There's Ludlow, who jumps around so much that she has to catch him up in midair. He broke one of his hind legs jumping over the sides of an older pen, just as his mother did, twice.

Lamson isn't the only pygmy rabbit worker who bonds with the animals. The day I visit Washington State, Becky Elias, the manager of the pygmy rabbit facility at WSU, is sporting a "Bunny Wars" T-shirt made to look like the opening credits of Star Wars. "Not so long ago, in a bunny pen far, far away," reads the front. "Help me bunny breeders, you're my only hope!" the back continues underneath a cartoon of a rabbit wearing a Darth Vader mask, followed by the names of all the WSU rabbits, some of whom are, yes, Yoda and Leia.

When Lamson, Onyx, and I arrive at the condor facility, Lamson and Kelli Walker, who lives there and manages it, start to make their rounds. As is true at all the pygmy rabbit facilities, we step into a shallow tub of disinfectant before entering the rabbit pen area. They weigh Sparrow, a 3-week-old rabbit. She was 106 grams the week before, and now she's 142. Her brother Mario has gained 31 grams over the same period, growing from 102.5 g to 133.5 g. Their sister Pippa's growth is even more impressive: 92.5 g to 130 g. Still, they all fit easily into Lamson and Walker's palms.

A moment later, it's time to set two adult rabbits, Bud and Poppy, up on a date, at "Chez Poppy." As the breeding season gets underway in January, males develop testes. Now, in April, Lamson and Walker are giving Bud and Poppy two or three nights together in Poppy's pen, and see what happens. As we watch Bud take his measure of Poppy's pen, Lamson points out that Bud is constantly marking sage and other material with the skin under his chin. She's convinced the rabbits have a scent gland there, and wants to prove it this summer with some careful observation.

Lamson leaves to prepare Onyx's new pen. She's brought sage from his Oregon Zoo pen, so the new space will smell familiar to him. She's also pulled out as much of the wild lupine, a poisonous plant his instincts may or may not tell him to avoid, as she could find. When the pen is ready, she goes back to her car and carries him to the pen, in a pet carrier designed for small cats. She then places him in his nest box, which actually is a small ice chest with a hole in the side of it.

ONE THAT DIDN'T MAKE IT: A bag containing the remains of one victim of the Northwest Trek outbreak, in pathologist Michael Garner's freezer.

"C'mon, Onyx, out of the box," she coaxes, urging him out of the new nest box through hollow corrugated plastic tubing that she and others use to mimic burrows. The tubing is connected to the hole in his box and runs under a mound of dirt, emptying out of the side of a mound covered in sage. When they first explore, rabbits visually memorize their runway, taking small steps, then returning to the nest, and gradually taking more steps. Onyx starts doing this as we watch.

"It's like rabbit heaven in here," says Walker as she starts snapping photos to document the release. Every new situation is a bit of an experiment, and Lamson wants to ask other pygmy rabbit experts what they think of the setup. Lamson squats patiently about six feet from Onyx's nest box, watching and encouraging him with clucks and coos. But by the time I leave, in the midafternoon, he hasn't emerged from the tubing, and Lamson will be staying awhile. "Nothing can get him in here, but I feel badly because of the rain," she says, looking up at a typical mostly cloudy Pacific Northwest sky. "We were hoping for a sunnier day."

When tissue samples of the first two dead Northwest Trek rabbits came into Northwest ZooPath, a pathology consulting service in Monroe, Wash., about 48 km northeast of Seattle, it wasn't clear that a serious problem existed. Founder Mike Garner referred them to his partner, John Trupkiewicz, who reads nonurgent slides. Trupkiewicz saw a cecal lesion, but didn't interpret it as cause for alarm, in line with the clinical evidence. After all, pygmy rabbits die all the time.

Suddenly, Northwest Trek lost five rabbits, and Garner and Case both realized something more serious was going on. Case tried bacterial cultures, which came up negative, and Garner confirmed those findings with special bacterial stains early in the investigation. "I was pretty much thinking a possible viral etiology with the first case I read, which was the third case in the series," says Garner. By then, he was getting samples from a new rabbit nearly every day from Northwest Trek. "By the time I received slides from the first one, I will have gotten an E-mail saying, 'I've lost two more.'"

In 2001 and 2002, veterinarians captured as many Columbia Basin rabbits in Washington as they could find. They only found 16.

Garner works out of a nondescript house-turned-office in Monroe. He's published about 120 scientific journal articles on animal pathology, and more than a half-dozen of the journals whose covers feature his studies grace a wall in his office. Every morning, some of the more than 80 zoos and 200 private clinics that ZooPath serves send him dead animals and flats of slides. The day I arrived in late April, the mail contained slides of two prairie dogs with a history of seizures, and two cobras showed up in a FedEx box sent by an anxious veterinarian in Westchester County, NY. Showing me around his garage, now converted into a necropsy room where he slices open dead animals, Garner predicted the snakes had died of Paramyxovirus infection. He turned out to be right.

Garner has done a large portion of the pathologic investigation for the pygmy rabbit community for years. He knows some of them, including Case, from working with other zoo collections. Others he knows from spending nine years at WSU in Pullman.

In March, Garner started to find lesions on the apical aspects of mucosal epithelium of the ileum and cecum in all the slides from dead pygmy rabbits, then went back to look at the first two cases, which were separated from the others by two weeks. He realized then it was the same disease, and he started thinking about viruses. "You can see the progression of the disease here," he says, pointing to a computerized slide of the large intestine of one of the dead rabbits. He shows me the progression of the infected cells: hypertrophy, followed by vacuolization, then necrosis. "This one here has become eosinophilic and has a pyknotic nucleus, and then they slough off into the lumen." Once the lamina propia becomes denuded, "these bacteria that are in the central lumen get into the bloodstream, causing sepsis, and that's the end of the road."

THE END OF THE ROAD? Pathologist Michael Garner points out features of a slide of the large intestine of a dead pygmy rabbit that made him suspect coronavirus was the cause of its sepsis. The progression is hypertrophy, followed by vacuolization, then necrosis. After cells slough off into the lumen, "these bacteria that are in the central lumen get into the bloodstream, causing sepsis, and that's the end of the road," says Garner.

The microscopic findings suggested a virulent coronavirus to him. WSU found coronavirus in the 2002-2004 outbreak, but concluded it was an incidental finding. Coronavirus lesions are subtle, says Garner, but Case's quick and careful sample preparation meant he had good tissue to work with for the 11 rabbits that eventually came his way. He and others stepped up their efforts, and Northwest Trek provided $1,000 to pay for additional tests.

Garner sent samples to the University of California, Davis, for transmission electron microscopy, to Michigan State University for immunohistochemistry and sequencing, to WSU for virus isolation, and to the University of Georgia for scanning electron microscopy. The $1,000 "is barely paying for their reagents," says Garner. "But being vets, or familiar with veterinary medicine, they're trying to do what they can. People want to study" the killer agent - and hopefully publish a paper on it, he says.

Although none of the Northwest Trek rabbits participated in the March 13 release, Ed Cleveland, the facility's head animal-care technician, took part in the ceremony at the Sagebrush Flat Wildlife Area. The pygmy rabbit community is close-knit, and other keepers and vets wanted to make sure Northwest Trek didn't feel it had failed in some way. "If anything, they were more successful at breeding in the beginning, with fewer problems" says WSU's Shipley. "We've all had our share of everything."

In the months before the release, the vets and colony managers shoveled out artificial burrows underneath sagebrush plants in the flats, about 100 meters apart, so rabbits would have their own territories, and then sealed the burrows to keep out weasels. They left some pellets and greens to give the rabbits a head start as they adapted to foraging full-time. Before the release, handlers had placed rabbits in larger prerelease pens to acclimate them to living in the wild. Some of these were tricks they learned when they went through the same process with Idaho rabbits on the grounds of the Idaho National Laboratory.

On the morning of April 13th, reporters, landowners, and others - all told, 45 people - gathered on the north end of the release area, totaling more than 3,000 acres. Two trucks, one each from the Oregon Zoo and from WSU, pulled up and crept about 100 yards farther into the area. Handlers then took all 20 rabbits in their individual pet carriers to their new homes. Each rabbit had a designated spot to discourage breeding by closely related rabbits.

Once the rabbits, wearing tiny necklaces with lightweight radios, were deposited in their artificial burrows, handlers left them to adjust to their new lives, but returned regularly to monitor the population, and kept an eye on the radio signals. As the rabbits spread out, that wasn't easy to do. "We ended up getting on a plane at one point," says Hays.

THE RABBIT WHISPERER: At a condor captive breeding facility, the Portland Zoo's Rachel Lamson prepares a large pen for a pygmy rabbit named Onyx, to prepare him for potential release into the wild. Lamson spends time with the rabbits seven days a week.

The pilot release has gone as well as expected, experts say. As of early May, five of the rabbits, two females and three males, were still alive. Two of the males, Grasshopper and his brother, Ant, were brought back to WSU to keep them safe from the predators that had presumably eaten the rest. The males, rebuffed as usual while the females were pregnant, ran off to find other females, unaware that there weren't any. Grasshopper managed to travel 10.5 kilometers. No litters have been seen so far, even though the mothers should be past the gestation period. Whether they gave birth and abandoned the litters, or didn't give birth at all, is unclear. "We know that there's a lot of interaction between the males and the females," says Hays, so there's potential for a new litter before July, when their breeding season ends.

The organizers have already learned some lessons. Hays says it may work better to release the females first, after they've given birth. "There are several possible ways to do that," he notes. "Hopefully, we will have improved next time. And if we're lucky and we have litters this spring, I think that will be fantastic, given this is our first attempt."

Even the handlers had to adjust to the rabbits' move to the wild, such as by replacing the released rabbits' names with numbers, says Hays. I ask Elias whether it was difficult to let the rabbits free, knowing their chances of survival were so small. She admits to weeping when the first five rabbits she raised died in captivity when she started working with them in 2002. But she wouldn't hesitate to release them into the wild. "That's where they belong," she says, even if "out there, they're food" for their typical predators.

"There was a lot of trepidation," says Hays. "We knew that the smaller the number of rabbits released, the smaller our chances of success. This was our first attempt, and people knew there were going to be a lot of them dying, but it was the right thing to do."

It's not just the people who get attached. The rabbits are often curious about humans, described in writings dating back to the 1940s, according to Elias. Such curiosity could, of course, instill too much trust and put them at risk of predators. "We're not raising them to go live in captivity," says Jim Evermann, a veterinarian at WSU who worked on the cheetah recovery project beginning in the 1980s. That project's goal was to keep an endangered species in captivity. "We're raising them to go back to the wild."

"We're anxious to get them back to Sagebrush Flats to breed with the females," says Elias, as we both watch Grasshopper - one of three males being considered for rerelease in the coming months.- devouring a long piece of grass in a small cage at WSU.

SPLENDID ISOLATION? Pygmy rabbits' habitat (shaded areas) is the Great Basin, starting at the Great Salt Lake and reaching northwest. Two distinct subpopulations, the Idaho and Columbia River subpopulations, probably became isolated by the Columbia River - which separates Oregon and Washington - thousands of years ago. There was much less suitable habitat for the Columbia River subpopulation, and they are now severely endangered.

What actually caused the sepsis that killed the rabbits at Northwest Trek is still unknown, although whatever it was stopped on March 15. There's always the possibility that electron microscopy might identify another agent, says Garner, but he's betting on a coronavirus. The Washington Animal Diagnostic Disease Laboratory at WSU found coronavirus particles when they did negative-stained electron microscopy. Unlike cows and pigs, there's no vaccine for coronavirus in rabbits.

Garner says that he still needs good evidence that the coronavirus is actually in the lesions, since it is omnipresent in the species. "If we're able to sequence the virus, that will govern how we approach the problem in the future," he says, noting that coronaviruses are notoriously hard to isolate. "If we're able to isolate it, the folks in Pullman will want to do infectivity studies, maybe in cottontails." Infecting an endangered species for an experiment is, of course, a nonstarter.

Two months after the outbreak started, things are basically back to normal at Northwest Trek. The rabbits left the makeshift hospital on April 7 and are living in their usual pens, while managers are carefully trying to breed them, since any contact can spread infection. Biosecurity measures remain high, but Case says that the Northwest Trek keepers already practiced very tight treatment protocols. "They're a little more watchful, maybe, for fecal consistency, but these are really things they know anyway. I can't say there have been any profound changes."

"Do I feel our medical support helped?" Case asks. "Yes, but on the other hand, the outbreak ran its course." Still, the rabbits are never quite out of the woods: The week before I visited Garner in Monroe, there were several cases of diarrhea in a group of Northwest Trek rabbits that had survived the outbreak. Garner is fairly sure it's a different disease process, since the diarrhea occurred more in the small intestine and was associated with fewer deaths. Still, he says, "If she loses any more, I want to see them."

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