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year ago this month, a group ofARS scientists and techniciansgave up their Christmas time offand even delayed family vaca-

tions to provide characterization of thefirst case of bovine spongiform enceph-alopathy (BSE)commonly called madcow diseaseto be found in the UnitedStates.

On December 23, 2003, a Canadiancow shipped to slaughter from a farm inMabton, Washington, had come up pre-sumptively positive for BSE in testing byUSDAs Animal and Plant Health In-spection Service (APHIS), which hasdiagnostic responsibility and regulatoryoversight for BSE issues. APHIS hadalready used the gold standard diag-nostic immunohistochemistry test, whichwas originally developed by ARS. Butfor the first U.S. case of BSE, APHISwanted additional scientific informationthat could be provided by the Westernblot test.

Disease-causing prions accumulate in thebrains of host animals. Here, veterinarymedical officers Robert Kunkle (left) andAmir Hamir examine prion distribution inbrain tissue of TSE-affected animals.

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In Pullman, Washington, ARS researchers developed the first practical live-animal test forscrapie, the TSE that afflicts sheep.

A

So APHIS put in a high-priority callto veterinary medical officer JuergenRicht and his colleagues at the Virus andPrion Diseases of Livestock Laboratory,which is part of ARSs National AnimalDisease Center (NADC) in Ames, Iowa.

We had experience with the Westernblot test and we had all the reagents onhand, explains Richt. So we put ourholiday plans on hold and got everythingready so that APHIS would have verifi-cation of the results from the immuno-histochemistry test.

On Christmas Eve, Richt and lab tech-nicians Semakaleng Lebepe-Mazur andDeborah Clouser provided APHIS witha report, 22 long hours after the samplesarrived in Ames. ARS veterinary medicalofficers Robert Kunkle and David Alt andtechnician Dennis Orcutt providedadditional DNA sequence information,confirming that the tissue samples actu-ally came from a cow and not a sheep,deer, or other animal.

Then on December 27, APHIS con-tacted Will Laegreid, animal healthresearch leader at ARSs U.S. MeatAnimal Research Center (MARC) inClay Center, Nebraska, to orchestrateDNA testing and analysis to trace theorigin of the BSE-positive cow. His grouphad previously developed bovine DNAmarkers for identifying animals thatcould be used for epidemiological trace-back. MARC teams worked around theclock preparing DNA samples. Late onNew Years Eve, after the last criticaltissues arrived, the processed sampleswere driven to the first of two indepen-dent, certified laboratories for geno-typing. Within days, MARC analysis ofDNA evidence confirmed the positivecow was of Canadian origin.

A Mysterious EnemyConducting such urgent testing is not

a usual part of ARSs work, but very littleis usual when it comes to the enigmatic

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TSEs Touch Off ARS Research

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A Western blot analysis done by techniciansDeborah Clouser (sitting) and SemakalengLebepe-Mazur was crucial in tracking thefirst U.S. BSE case.

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Histotechnologist Jean Donald prepares5-micrometer-thick sections of tissuecollected from TSE-affected animals. Thesections are then mounted on glass slides,stained, and examined by pathologists.

PEGGY GREB (K11646-1)

class of animal diseases called trans-missible spongiform encephalopathies(TSEs). These diseases are caused byabnormal prions.

Normal cellular prion proteins occurnaturally in many tissues, including brainand other nerve tissue, but their functionsare not well understood. These normalprion proteins can change and aggregateto form disease-causing prions.

The prevailing theory is that prionschange their shape and fold into an ab-normal form that accumulates in the brainand causes lesions. If the abnormal prionsare transmitted from an afflicted animalto a new host, they may cause the newhosts prions to begin folding abnormally.

Discovery of these prion traits hasaltered the accepted scientific groundrules for what can cause disease. Prionsdo not contain DNA or RNA as do fungi,bacteria, viruses, viroids, or any otherpreviously known infectious entities.They are simply proteins, and proteins

had not been believed to be infectious ontheir own.

BSE itself is a fairly new disease; itwas first diagnosed in 1986 in GreatBritain. The disease has cost theEuropean Union livestock industry atleast $107 billion as of this writing.USDA has maintained an aggressiveimport exclusion and surveillanceprogram since 1986 to minimize thespread of BSE. As of this date, only oneimported BSE case has been found in theUnited States.

Three other animal prion diseases areknown today: Scrapie, which affectssheep and goats, was first recognized inGreat Britain more than 250 years ago.The disease did not appear in the UnitedStates until 1947, when it was found in aMichigan flock. Transmissible mink en-cephalopathy (TME) is a rare illness thataffects mink. It too was first detected inthe United States in 1947, on a mink

ranch in Wisconsin, and on ranches inMinnesota and Idaho in the 1960s.Epidemiologic data from these outbreakstrace the cases to one common purchasedfood source. Since then, TME outbreakshave also been reported in Canada,Finland, Germany, and the republics ofthe former Soviet Union.

Chronic wasting disease (CWD) is aTSE of deer and elk. CWD has been re-ported in free-ranging mule deer, white-tailed deer, and Rocky Mountain elk inColorado, Wyoming, South Dakota, NewMexico, Utah, Wisconsin, Nebraska, andIllinois; and in game-raised elk in SouthDakota, Kansas, Montana, Oklahoma,Colorado, Nebraska, Minnesota, andWisconsin. The disease has also beenfound in game-raised elk and a few free-ranging deer in Canada.

ARS has one of the worlds most com-prehensive research programs investigat-ing TSEs. It is the only organization

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Chemist Chris Silva (left) and research leader J. Mark Carter load samples for analysis viananospray liquid chromatography coupled to mass spectroscopy. This state-of-the-arttechnology characterizes BSE prions with unprecedented precision.

At the ARS National Animal Disease Center in Ames, Iowa, animal caretaker Gary Hansentends to two jersey steers. The steers are used as controls in a CWD cross-speciestransmission experiment in which cattle were inoculated intracerebrally with CWD-infectedbrain tissue.

PEGGY GREB (K11654-1)

studying all four TSEs in animals. ARSis taking a very integrated approach toTSE research, with collaborative projectsinvolving many disciplines and scientists.While each TSE is unique in many re-spects, there is so much to learn aboutprion diseases that what researchers learnabout one TSE may give insight intoanother.

DiagnosticsNow-retired ARS veterinarian Janice

Miller developed the first immunohis-tochemistry method for diagnosis ofscrapie in sheep in 1993. This test wasmuch more specific and less burdensomethan any other at that time. In 1998, ARSmicrobiologist Katherine I. ORourke atthe Animal Disease Research Unit inPullman, Washington, further increasedthe tests specificity and ease of use byincorporating monoclonal antibodies.Use of these monoclonal antibody re-agents was then broadened to be able todiagnose the other TSEs.

Later, ORourke had a real break-through when she discovered that prionscollect in pockets of lymphoid tissue ina sheeps nictitating membrane, or thirdeyelid. A veterinarian can take a sampleof the tissue with only a local anesthetic,which meant that there was finally a prac-tical, live-animal test for scrapie. Thislive-animal test is now an approved diag-nostic test for scrapie in the United States.

A very rapid, ultra-sensitive test thatcould be used before animals show anysymptoms, especially with BSE in cattleand CWD in deer and elk, is still a majorresearch goal.

One approach being taken today byARS chemist Bruce C. Onisko at theFoodborne Contaminants Research Unitin Albany, California, is use of mass spec-trometry to identify extremely low lev-els of prions. Mass spectrometry revealsstructural information from biologicalcompounds by ionizing a molecule ofinterest, fragmenting it by collisions withan inert gas, and then applying mass anal-ysis to the fragmentation products.

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Antibodies only let us find prions inamounts greater than 1 picomole. Forlive-animal testing we need to be able toreliably and quantitatively detect concen-trations 3 to 4 orders of magnitude lessfrom easily obtainable tissues, explainsOnisko. And we need to be sure we arelooking at only the abnormally config-ured prion protein.

Such a sensitive test would helpdiagnose animals with abnormal prionsbefore they start showing clinical symp-toms. ARS has now applied for a patentfor a new diagnostic test based on thistechnology.

ARS chemist Christopher J. Silva, alsoat the Foodborne Contaminants ResearchUnit, is using mass spectrometry to de-velop a way to test feeds for the pres-ence of animal materials.

A test for the presence of prions inanimal feed is problematic. Epidemiolo-gists in the United Kingdom showed thatprions are not evenly distributed in ani-mal feed, so an analytical sample mightnot be representative of the whole feedlot. Furthermore, could such a test be sen-sitive enough to detect rendered prions?

Instead, Silvas work on detecting thepresence of prohibited animal materialsin animal feeds would serve as an im-porta