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by Charlie Powell In 1947, a flock of sheep was legally imported into the United States from Canada after a routine sale. The Canadian seller had developed his flock by adding sheep from flocks in the British Isles for several years prior to the sale. Soon after the new flock’s arrival in Michigan, the animals started displaying peculiar clinical signs of disease. At first, there were behavioral changes subtle enough that only an experienced shepherd might notice. Over time the signs progressed to scratching and rubbing against fixed objects, apparently to relieve itching. Some of the sheep showed a loss of coordination and weight loss. However, the animals kept a good appetite. Signs soon worsened from the unusual to the bizarre. Some animals bit their feet and limbs, smacked their lips, and showed gait abnormalities. The gait problems were characterized by high stepping of the forelegs, bunny hopping, and swaying of the rump. When the animals were left alone, they appeared normal. But any sudden noise, movement, or handling stress caused some to fall over and tremble in a convulsive-like state. Veterinarians who were called in to see the Michigan sheep ruled out progressive pneumonia, listeriosis, rabies, pregnancy toxemia, toxins, and external parasites as the cause of the problems. Before long it became apparent that the U.S. had its first case of scrapie. By 1952, the secretary of agriculture would be forced to declare a state of emergency in an attempt to eradicate scrapie in the U.S. It wasn’t until April 1998—51 years after the first outbreak—that USDA and Washington State University veterinary researchers announced that they had developed a practical pre-clinical test for scrapie. Before the discovery, shepherds of various breeds, including the popular Suffolks, and a number of crossbreeds, simply had to wait—and hope—that their sheep would not display the characteristic signs of scrapie that would require their flocks to be destroyed. “I always told producers that there were three keys to controlling scrapie in the U.S.,” says Katherine O’Rourke, the USDA Agricultural Research Service microbiologist and scrapie research team leader at WSU. “There was the genetic selection to avoid susceptible breeds and lineages; the field epidemiology that allows one to avoid infected flocks and eradicate those that are; and a high-quality, practical live animal test that would detect the disease before the signs appeared and the whole flock was considered infected. For years we had two of the three keys but lacked the live animal test.” The urgency to eradicate scrapie becomes clearer when one understands a bit more about this family of diseases. Scrapie is a type of transmissible spongiform encephalopathy (TSE) similar to the so-called mad cow disease that has devastated the European beef industry. It has been recognized as a disease of sheep in Great Britain and other countries in Western Europe for almost three centuries. The agent that causes the disease is thought to be a small infectious protein particle called a prion. Scrapie is also related to the fatal human spongiform encephalopathy known as Creutzfeldt-Jacob disease. This family of fatal degenerative diseases of the central nervous system occurs naturally in other species, including deer, elk, mink, and cats, as well as sheep and goats. “Spongiform” refers to the common postmortem pathology that is common to all TSEs and remains the only definitive diagnosis for the diseases. At autopsy, sectioning of the brain of infected animals and people shows characteristic spaces in the brain not unlike those in a household sponge. While there is still some debate as to whether scrapie and other TSEs are caused by prions or whether prions are the byproduct of an undetermined infectious agent, scientists agree that the proteins are at least a sentinel. Virtually all species of animals, including humans, have prions and the genetic sequences that code for them in their body naturally. Current theories suggest that for whatever reason, a normal prion will fold and change shape and itself become an infectious agent without the benefit of any nucleic acids.  Once a prion changes shape to the pathologic form, it is designated as PrP. A PrP is thought to be able to change other normal prion proteins into PrP simply through contact. The significance of this is that for the first time in medical history, infectious disease is being caused by a simple protein that is normally present and without the benefit of replication or reproduction, unlike other infectious agents like bacteria or viruses. Further danger to public health comes from the fact that PrPs are extremely resistant to normal enzyme breakdown, heat, and sterilization processes. Under certain conditions, PrPs have even been shown to survive incineration. Currently there are thought to be at least two cases of human TSEs that were transmitted by surgical instruments that had been cleaned and sterilized. In sheep, scrapie is thought to be spread most commonly from ewe to offspring and other lambs in lambing groups through contact with the placenta or placental fluids. “At WSU we were assembling a scrapie flock for study, and we needed these condemned sheep,” says O’Rourke. “It was hard to travel out to these small farm flocks only to see the people and the animals they cared about so devastated. In one instance, the sheep were part of a 4-H project, and the sale of the animals was going to help provide college money. It was a great incentive to continue on.” In the laboratory, WSU and USDA scientists were busy trying to develop antibodies to the isolated prions. The difficulty was that the normal prion is present in virtually all animals studied, so their immune systems do not recognize it as foreign. Without this recognition, antibodies are not formed against it. But over the years, immunologists used a variety of common techniques to develop the immunohistochemicals necessary for labeling abnormal prions in tissue sections. “We didn’t do anything that is not commonly done in similar laboratories worldwide,” explains Tim Baszler, a veterinary immunologist and associate professor in the WSU College of Veterinary Medicine, “but it was very difficult to develop the reagents.” What Baszler and his coworkers did do is painstakingly examine each blood sample in each trial. Most were negative, and some researchers might have stopped there or begun only surveying representative samples from each experiment. Eventually, reactivity to the PrP was discovered, but at a low level and in only a few animals. Once it was found, however, building the rest of the reagents for a test was relatively easy. Now came the challenge of finding PrP in living tissue. A 1996 paper by a research group in the Netherlands described a preclinical test that used a biopsy of the lymphoid tissue in sheep tonsils. WSU’s research team tried that too. They found that getting a biopsy from a sheep’s tonsil requires general anesthesia and is hardly practical as a test for eradication purposes. But they also learned that Baszler’s reagents worked in sheep. Once the tissue was obtained, fixed in formalin, and sectioned, it could be treated with Baszler’s specific monoclonal antibody that contained a second antibody labeled with a common visual dye called a chromagen. Normal lymphoid tissue complete with normal prions comes out a pale shade of blue. Lymphoid tissue from sheep with scrapie appears blue, but the reagents bind an unmistakable red dye to the PrP concentrations. But for some unknown reason, researchers around the world have discovered that not all tissues concentrate the PrPs alike. By 1997, all the elements for developing a pre-clinical test except one were in place at WSU. “We just needed to find a good source of the prions in living tissue that was easily accessible,” O’Rourke says. “We needed a real veterinarian to help us.” The scrapie research group soon invited Steve Parish, a 1973 WSU veterinary alumnus, to join their weekly meetings. Parish heads up the large animal section of the WSU Veterinary Teaching Hospital. “The agricultural animal clinical group has a long history of collaborating with the college’s basic science researchers,” says Parish. “So there I was in the first two or three of these meetings, and I was surrounded by all these Ph.D.s listening to the problem they had finding the right tissue to biopsy around the head or neck in sheep. I certainly didn’t understand all they were talking about with prions and immunohistochemicals and such, but I knew the animals, and it was just like a bell went off. I asked, ‘Have you tried the third eyelid?’ The room kind of went quiet, and they all turned and looked at me with this blank look, and we knew.” The basic anatomy of a sheep’s third eyelid, or nictitating membrane, includes two clusters of lymphoid tissue. Under Parish’s direction, a drop of local anesthesia and a small biopsy provided ample tissue. Within days, with the help of veterinary students, the research team had gathered a number of biopsies from normal and infected sheep. Now the work fell to Baszler and colleagues again. Would there be enough prion in the tissue? Would the reagents work on these sections? “The results were so good, they were a bit startling,” says Baszler. “I just happened to step out of my lab after looking at sections, and Steve [Parish] was walking down the hall, so I asked him to come in. I set up two slides and asked him to tell me which was the infected tissue and which wasn’t. He took one look and then gave me this big smile.” By the spring of 1998, WSU’s scrapie research team had their work accepted for publication and the reagents and techniques securely locked up in patent acquisition. In June, the team traveled to Washington, D.C. to receive the USDA’s Honor Award for personal and professional excellence. “This is a remarkable scientific achievement by a dedicated core of scientists working together,” says Terry McElwain, interim dean of the WSU veterinary college. “Our hope now is to perform the necessary scientific validations and be able to use technology transfer to the private sector for distribution of an effective, inexpensive scrapie test to the sheep producers of the world.” For all its promise though, O’Rourke says the test needs a rigorous validation before it can be made commercially available. “We need to know how sensitive the test is and how specific it is, because no test is 100 percent sensitive and specific. By that I mean we know we will undoubtedly get false negative tests and false positive tests. What percentage of those we get will tell us how good our test is.” Already the WSU work is spreading worldwide. Laboratories are finding the reagents developed in Pullman are very sound and may have applications in other areas of diagnosis. A Belgian team has demonstrated that the antibody system will bind to PrPs in human brain tissue. The WSU team is currently considering testing the sheep reagents on the TSE that occurs naturally in deer and elk, too. “We also need to know how soon we can identify an infected sheep and eliminate it,” says O’Rourke. “Right now we’ve been able to detect PrPs in 14-month-old sheep.” For Parish personally, the award and accolades pale in light of the accomplishment for the public and the spirit of cooperation it took to make it happen. “This is scientific and clinical collaboration at its best,” he says. “For 23 years since I’ve been here, we’ve never failed to help the scientists in the lab with what we see in the clinic and vice versa. Identifying a tissue like the third eyelid goes back to my original training as a veterinary student here at WSU. Dr. Gary Bryan, our recently retired veterinary ophthalmologist, taught me there was lymphatic tissue there more than 25 years ago. Developing a test like this is being proactive in consumer protection. I’m proud to have been a part of it.” Charlie Powell is information coordinator for the College of Veterinary Medicine. |
Before the discovery, shepherds of various breeds, including the popular Suffolks, and a number of crossbreeds, simply had to wait—and hope— that their sheep would not display the characteristic signs of scrapie that would require their flocks to be destroyed. 
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