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From Agricultural Research magazine, December 1995

Alcatraz for Animal Disease

The Plum Island laboratory keeps foot-and-mouth disease at bay.

The bad news came first from Baltimore: "A disease has made its appearance among the horned cattle in our neighborhood, which threatens us with very serious inconvenience."

That issue of "American Farmer," published in Baltimore on a steamy July day in 1825, went on to report that bewildered farmers were finding their animals lame, the fleshy part in the division of the hoof "sore entirely through," and, worse still, "the disease appears to be contagious."

For the first time, foot-and-mouth disease (FMD) in the United States had officially made the news.

 
Unfortunately for livestock producers, foot-and-mouth disease is still a "hot story" 170 years later. As recently as June of this year, a suspicious livestock disease outbreak in Arizona and New Mexico had federal and private veterinarians scurrying for confirmation that the problem at hand was not the foot-and-mouth virus, but its copycat cousin, vesicular stomatitis.
Aerial view of PIADCThe Plum Island Animal Disease Center stands ready to defend against foreign animal diseases. It is home to a foot-and-mouth disease vaccine bank supported by Canada, Mexico, and the United States. Photo by Keith Weller (K6086-7)

[Note: PIADC land and facilities were transferred to the Department of Homeland Security in June 2003.]

"Foot-and-mouth disease is the most economically important single livestock disease worldwide and perhaps the most contagious," explains Harley W. Moon, director of USDA's Plum Island Animal Disease Center (PIADC) in New York. "It can spread by air to a broad number of cloven-hooved species, infecting animals from cattle and swine to wildlife."

Indeed, says Moon, by the time Baltimore's "American Farmer" picked up the story, it was old news: Farmers have feared foot-and-mouth disease for more than four centuries. It is so dreaded in this country that even though the United States has not had an outbreak since 1929, Congress passed legislation in the 1950's that made it illegal to possess live foot-and-mouth virus on the U.S. mainland—even in the form of protective vaccines.

Moon stands at the center of what could be called FMD Central, because that post-World War II law has made the Plum Island center the only place in the United States where FMD can be studied. Although PIADC is officially part of Suffolk County, New York, it is set on an 840-acre island about 1.5 miles northeast of Orient Point, New York, and 12 miles southwest of New London, Connecticut. It is separated from the mainland by Long Island Sound to the north, Block Island Sound to the south, and Plum Gut to the west.

A small army of scientists braves the waves of Long Island Sound every weekday morning to reach their island laboratories, where they search for new weapons against this livestock disease that threatens no consumer's health, but every consumer's pocketbook.

According to a 1983 study by the National Research Council's Subcommittee on Research and Diagnosis of Foreign Animal Disease, a "modest" outbreak of FMD in this country would cost $54 million to control. Other studies have put that total as high as $690 million. But the overall economic burden, says Moon, would be many times heavier.

"Last year, the United States exported nearly $4 billion worth of beef," he notes. "If we had an FMD outbreak in the United States today, our exports of beef would be halted for at least a year. A lot of our beef customers overseas are in countries that are FMD-free, and they wouldn't want to take the chance of bringing the virus into their countries through our meat products.

 
Biologist  examines cell cultures
Biologist Barry Baxt examines cultures of living cells used to assess the effects of genetically engineered mutations on virus adsorption. (K6094-3)

"Even countries that have FMD, but have it under control to some extent, could buy their beef elsewhere and not complicate matters. We would have to stay FMD-free for a year before we could sell again overseas; that's the internationally agreed-upon custom."

Moon says that if the United States were not successful in ridding itself of FMD immediately but allowed the infection to spread through the country, the cost over 10 years' time could run to $30 billion in lost sales and production—a total he describes as "very conservative."

Standing between U.S. livestock producers and consumers and this economic disaster are two Plum Island entities: the Agricultural Research Service's Foot-and-Mouth Disease Research Unit and the Foreign Animal Disease Diagnostic Laboratory operated by USDA's Animal and Plant Health Inspection Service (APHIS).

What FMD Does, Physiologically

Perhaps surprisingly, an animal that contracts FMD may not die, although it will suffer some agonizing days. The FMD virus causes blisters known as vesicles to form on the animal's lips, gums, and tongue, as well as in the flesh adjoining the hard part of its hooves. These vesicles can be caused by a handful of diseases—including FMD, vesicular stomatitis, swine vesicular disease, and vesicular exanthema—known collectively as vesicular diseases.

Soon the tops of the blisters tear off, leaving enormous raw areas that make it almost impossible for the animal to eat, drink, or walk. Even though the animal is likely to recover, the livestock producer's worries are hardly over, notes microbiologist Cecelia A. Whetstone, head of the PIADC Foot-and-Mouth Disease Research Unit.

"If an animal has been infected, even though it survives the disease, it can still carry the virus and spread it to other animals," says Whetstone. "And it’s very important to distinguish between an animal that's been infected and one that's been vaccinated."

But that can also be very difficult—and that's where Fred Brown comes in. An ARS microbiologist with a lifetime of experience studying FMD, Brown worked with APHIS' Juan Lubroth to pinpoint a protein in the FMD virus that's present in the virus' early days of self-reproduction, but not in the final virus particle.

"We can grow the virus in tissue culture, and this particular protein stays behind in the culture cells when you extract the virus," explains Brown. "So you can make a vaccine from that virus, inject it into livestock, and the animals' immune systems won't make antibodies against that particular protein because it wasn't present in the vaccine.

"But if an animal has been infected with FMD virus, that protein will have been present, and you will find antibodies against it in the blood. So, to differentiate between an animal that's been vaccinated and one that was actually infected with FMD virus, you could run tests to check for antibodies against that indicator protein."

Brown and fellow ARS microbiologist John F.E. Newman are also looking at the creation of a vaccine that uses only a string of some 20 amino acids from the virus—just enough of a taste of the virus to jump-start the injected animal's immune system into producing enough antibodies to neutralize the virus. This prototype vaccine has effectively protected cattle against FMD in field tests in Argentina, Brown says.

 
Microbiologist examines sequencing gels
Genetically engineered mutations inserted into the foot-and-mouth disease virus genome show up on sequencing gels examined by microbiologist Elizabeth Reider. (K6093-8)

ARS microbiologist Barry Baxt is also no newcomer to the FMD battle. He's spent nearly two decades looking for the answer to how FMD virus takes hold in an animal, based on the idea that if there's no attachment, there's no infection.

"It's been known for years that when animals make antibodies against FMD virus, most often those antibodies are in response to small areas in one of four structural proteins on the virus particle," says Baxt. "The structural protein in question is known as VP1."

In every foot-and-mouth virus for which the genetic code has been mapped, a little three-amino-acid group appears in the same place on VP1. Scientists call this amino acid group RGD—a bit of acronymic whimsy because the three amino acids are arginine, which sounds as if it should begin with the letter "R"; glycine; and aspartic acid, which is typically pronounced as though it were "as-par-dic," contributing the letter "D."

"This same RGD sequence is also found on a number of proteins that are involved with how all sorts of cells attach to each other," says Baxt. "Eventually this led us to suspect RGD was involved in the virus being able to bind to cells in the animal."

In tests at Plum Island, Baxt and co-workers made strings of amino acids—called peptides—that mimicked the RGD sequence. They then took cells that were susceptible to FMD infection and mixed them with their synthetic peptides and also with actual FMD virus that had been radioactively labeled so its presence could be easily detected.

The synthetic RGD peptides were able to elbow out the virus and bind to the cells with 50 to 70 percent efficiency, Baxt reported. But when the scientists changed the RGD makeup in their manmade peptides by only a molecule or two, the peptides weren't able to crowd the virus out.

"This showed RGD is obviously involved in the virus' ability to bind," notes Baxt.

He and fellow ARS microbiologist Peter W. Mason saw a tempting chink in the FMD virus' formidable armor. What if, they mused, you made an FMD vaccine in which the virus' RGD sequence had been deleted? That could mean the virus wouldn't likely cause disease because it couldn't bind in the animal. But its mere presence in the vaccine would signal the animal's immune system to start producing protective antibodies.

Mason and ARS microbiologist A. Elizabeth Reider took the genetic material of FMD virus through a series of complex manipulations. They ultimately produced what is known as copy DNA (cDNA) of the virus, but minus the crucial RGD amino acid sequence.

Next, they grew the cDNA in a non-disease-causing bacteria, a procedure that can produce relatively large amounts of cDNA.

From the multiplied cDNA, the scientists produced RNA—the part of the virus' inner mechanisms that enables it to reproduce itself—and put that into living cells to produce live FMD virus that's incapable of attaching to cells and causing infection in an animal.

The test came in the summer of 1994, when ARS veterinarian Thomas St. C. McKenna vaccinated three steers at Plum Island with the prototype RGD-deleted foot-and-mouth vaccine. Three other steers were vaccinated with traditional FMD vaccines made from live virus, and another three steers received no FMD vaccines.

After a month, McKenna reports, blood tests of the six vaccinated steers showed their immune systems had produced antibodies to fight FMD virus, while no antibodies against FMD were seen in the nonvaccinated animals. Then the steers were all placed in a room for 2 days with an FMD-infected pig.

"Within a week, the nonvaccinated steers all got foot-and-mouth disease," reports McKenna. "The six vaccinated animals, including the ones with our RGD-deleted vaccine, remained clinically normal, with no disease at all.

  Microbiologist John Newman uses an x-ray generator to determine the particle structure of foot- and-mouth disease virus. This knowledge will be used in developing new vaccines. (K6096-11) Microbiologist uses x-ray generator

"I think the RGD-deleted vaccine worked at least as well as the standard inactivated virus vaccine, but with a big advantage: You could use it in this country," McKenna continues. "It's true that the standard inactivated vaccine is technically a killed virus, but it was still originally live virus that's been inactivated."

Indeed, the standard inactivated virus vaccine could be a recipe for disaster, says Peter Mason.

"Each dose of the current inactivated vaccine originally contained greater than 10 million cattle-infectious doses of the live FMD virus," he points out. "So if the inactivation is incomplete in any way and 200 million doses are sold every year worldwide—you can imagine what could happen. It's been well-documented that vaccine production plants and vaccines can be the source of FMD outbreaks, although that has been less of a problem recently."

ARS microbiologist Marvin J. Grubman pursues another approach to FMD control at Plum Island. "We've been studying a particular gene in the FMD virus whose protein product has the capability of cutting apart a protein in the animal cell that the virus wants to take over," explains Grubman. To understand what's happening, one might think of the animal's cell—known as the host cell—as a factory.

The crucial host-cell protein fuels the factory machinery and directs it to produce more protein needed by the animal. But if the virus protein, called leader protein, can disable the host-cell protein by splitting it apart, the virus can then commandeer the factory machinery to make the product it most desires: more disease-causing virus.

"Since the leader protein works by cleaving the host-cell protein, what happens if the host-cell protein is not cleaved?" asks Grubman. "The virus then has to compete for the use of the cell's mechanisms, and it doesn't grow as quickly.

"In a live animal, when this happens, the virus grows so slowly that by the time it comes out of the cell, the animal's immune system has already produced antibodies against it and won't let the virus spread to cells where it can cause damage."

In tests at PIADC, Grubman and ARS microbiologist Maria E. Piccone grew FMD virus minus its leader protein gene in tissue culture. Then Thomas McKenna inoculated a cow with 3 million of the theoretically infectious virus particles, all minus their leader protein. When the cow's blood was subsequently checked, it contained antibodies against foot-and-mouth virus, but the cow showed no signs of disease.

"By comparison, when we inoculated a cow with just five infectious virus particles of the complete parent virus, that cow got sick and had lesions," Grubman said. "Since this vaccine made of virus minus its leader protein still uses live virus, it couldn't be used in the United States. But it could be used in countries that have FMD."

According to ARS' Fred Brown, there are still plenty of such countries. For example, foot-and-mouth disease is known to be present in Colombia, Brazil, and India and in African countries.

 
Veterinarian examines tissue from animal suspected of having FMD
At the Plum Island Animal Disease Center, veterinarian Alfonso Torres examines tissue from an animal that was suspected of having foot-and-mouth disease. (K7028-2)

Happily, Canada and Mexico are now FMD-free. But Mexico had a severe outbreak as recently as the late 1940's and early 1950's. That outbreak prompted Congress to approve establishment of PIADC in 1954, when USDA acquired the island that was formerly Fort Terry from the U.S. Army.

For all its geographic isolation, PIADC maintains extensive ties with the U.S. mainland—as well as other countries.

"If a veterinarian anywhere in the United States sees a suspicious vesicular disease, as happened in Arizona and New Mexico the past summer, they send samples from the animal—preferably pieces of blister and fluid—to Plum Island for diagnosis," says Charles A. Mebus, who retired in June as head of APHIS' Foreign Animal Disease Diagnostic Laboratory at PIADC.

"We also teach five 1-week foreign animal disease courses a year at Plum Island," he says. "This includes two for APHIS field veterinarians, one for people who teach about foreign animal diseases in veterinary colleges and for people from state diagnostic labs, one for second-year veterinary students, and one for veterinarians from Agriculture Canada."

Plum Island is also home to an FMD vaccine bank of up to 2 million doses for each of the seven serotypes of FMD. The doses, kept in concentrated form, are stored at -70o C or in vaporized liquid nitrogen. While the doses will keep for a decade or more, Jim House of APHIS' foreign animal disease lab checks their viability regularly.

"This vaccine bank is for the United States, Canada, and Mexico," explains Mebus. "Each country contributes to its upkeep and helps decide what should be in there. The idea is that if they were needed, these vaccines could be brought into the field in a relatively short period of time."

Better Diagnostic Tools Are Critical

APHIS veterinary medical officer Alfonso Torres says foot-and-mouth diagnostic needs today are twofold. Torres is chief of PIADC’s Foreign Animal Disease Diagnostic Laboratory.

"We need to be able to do a quick, accurate diagnosis of any vesicular disease in the United States, including foot-and-mouth disease," he says. "And when animals are imported from foreign countries, we need to be able to test for antibodies as a measurement of whether they've been infected with foot-and-mouth disease."

APHIS is the bulwark between U.S. livestock and imported diseases, holding in quarantine animals imported from FMD-infected countries and testing them repeatedly before releasing them in the United States.

 

"The vaccines now available don't prevent an animal from getting infected; they just prevent that animal from getting sick," Torres explains.

"Vaccinated animals that get infected later can be long-time carriers of the disease. So when we import animals now, we exclude any animal that has antibodies against FMD virus. But if we had a test to distinguish between vaccinated and infected animals, that would be very useful."

All these demands keep the Plum Island team coming back day after day, looking for answers to the centuries-old problem of FMD.

"Our goal is a vaccine that's safe to produce and use in an FMD-free area," says ARS' Whetstone. "Even though FMD is a foreign animal disease, it's potentially the single greatest threat to livestock in American agriculture.

"We need better control strategies, better diagnostics that can be used on the farm, and vaccines that can be made and used anywhere in the world. Our group here at PIADC is focusing on that in collaboration with research groups around the world. We're producing first-rate science to meet an urgent need of agriculture."—By Sandy Miller Hays, ARS.

All of the scientists mentioned in this article can be contacted at the Plum Island Animal Disease Center, P.O. Box 848, Greenport, NY 11944; phone (516) 323-2500, fax (516) 323-2507.


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