Advancing Prion Science: Guidance for the National Prion Research Program (2004)

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_ ~ Surveillance for Transmissible ;_ Spongiform Encephalopathies in the United States Comprehensive surveillance for transmissible spongiform encephalo- pathies (TSEs) in people and animals in the United States is in the best interest of public health and the economy. A number of na- tional and state programs are conducting limited surveillance of both ani- mals and humans. This chapter reviews present TSE surveillance programs and recommends strategies to improve and expand them. A new human TSE, variant Creutzfel~t-Takob disease (vCTD), was iden- tified in the United Kingdom in 1996 (Will et al., 1996) (see Chapter 21. Evidence that eating beef products containing the infectious agent of bovine spongiform encephalopathy (BSE) causes vCTD in humans (Coulthart and Cashman, 2001) has led many countries, including the United States, to conduct surveillance for both vCTD and BSE and to try to prevent the entry or spread of BSE within their borders. To date, no case of BSEi and no endogenous cases of vCTD have been detected in the United States. Although this country appears to be at low risk for these two TSEs (HCRA and TUCCE, 2001), the evidence that the infectious agent of BSE crossed the species barrier into humans has generated considerable concern about a uniquely North American TSE of animals chronic wasting disease (CWD). This fatal illness of deer and elk (described in Chapter 2) appears to have spread from its original location in Colorado and Wyoming to 10 more states and two Canadian provinces since the mid-199Os. The U.S. Depart- ment of Agriculture (USDA) declared a state of emergency in 2001 after a iEDITORS' NOTE: After this report was completed, the first U.S. case of BSE was identi- fied in Washington State and was announced to the public on December 23, 2003. 125

126 ADVANCING PRION SCIENCE CWD-infected cervid was discovered east of the Mississippi for the first time (USDA Office of the Secretary, 20011. Now, virtually every state has or is creating a CWD surveillance program. For these reasons, the committee believes more surveillance and epide- miological research should be conducted to ensure the early detection of a new human TSE, should one emerge; to monitor scrapie, the likely cause of BSE; and to monitor the spread of CWD. The first two sections of this chapter describe the surveillance programs in place to detect human and animal TSEs in the United States; included are recommendations for re- search that would strengthen these programs. The final section presents the committee's recommendations for research into the epidemiology and natu- ral history of TSEs; discoveries in these largely uncharted waters would improve U.S. capabilities to conduct surveillance for TSEs. U.S.SURVEILLA~4CE FOR HU MARE TSEs U.S. surveillance of human TSEs relies almost exclusively on mortality data because there is no uniform system for reporting human cases of these diseases as they are diagnosed. Only 12 states have made Creutzfel~t-Takob disease (CTD) a notifiable disease. Nevertheless, mortality data for human TSEs are a reasonable surrogate for incidence data because these diseases are uniformly fatal and have a relatively short clinical phase about 4 to 17 months. The United States conducts nationwide surveillance for human TSEs and studies their epidemiology through two principal organizations: the Centers for Disease Control and Prevention (CDC) in Atlanta and the Na- tional Prion Disease Pathology Surveillance Center (NPDPSC) at Case West- ern Reserve University in Cleveland, Ohio. CDC funds NPDPSC. Three forms of human TSEs are known to occur in the United States. The most common of these occurs spontaneously as the result of an un- known cause. Sporadic Creutzfel~t-Takob disease (sCTD) and sporadic fatal insomnia (sFI) fall into this category. The second form is due to mutations in the gene that codes for a prion, the protein thought to be the infectious agent of TSEs (see Chapter 21. The more than 50 distinct subtypes of this genetic form of human TSE are called by one of three names: familial Creutzfel~t-Takob disease, fatal familial insomnia (FFI), and Gerstmann- Straussler-Scheinker disease. The third and least common form of human TSE in the United States is due to transmission of the infectious agent through transplanted tissue or tissue extracts from an individual with a TSE or from surgical instruments used on a TSE patient.2 This illness is called iatrogenic Creutzfel~t-Takob disease (iCTD). 2vCJD is also infectious, but no endogenous cases of the disease have appeared in the United States.

SURVEILLANCE FOR TSEs IN THE UNITED STATES latrogenic/l nfectious Genetic '0 5% ~ ~ pi \ 127 Sporadic ~88% FIGURE 6-1 The relative occurrence of sporadic, genetic, and iatrogenic forms of human TSEs in the United States, 1997-2002. SOURCES: National Prion Disease Pathology Surveillance Center (NPDPSC) (2003~; personal communication, P. Gambetti, NPDPSC, May 2003. Table 2-1 in Chapter 2 provides some detail on each of these diseases, all of which are rare and fatal. Of the nearly 600 U.S. cases of human prion disease examined neuropathologically from 1997 through 2002, the spo- radic forms accounted for approximately 88 percent of cases (the vast ma- jority being C]D), three genetic forms accounted for approximately 12 per- cent, and iC]D accounted for less than 0.5 percent, as shown in Figure 6-1 (NPDPSC, 2003; personal communication, P. Gambetti, NPDPSC, May 2003~. Through an analysis of mortality data, CDC determined that the rate and characteristics of human TSE deaths identified in the United States between 1979 and 2000 were stable over time and were consistent with the internationally accepted mortality associated with the disease (Belay and Schonberger, 2002~. The average annual age-adjusted TSE death rate in the United States from 1979 to 2000 was 1.07 cases per 1 million population (Belay, 2003), close to the worldwide rough annual average of 1 case per 1 million population. This finding suggests that endogenous vC]D has not occurred in the United States. A comparison of the age distribution of the United Kingdom's vC]D cases with that of the United States' sC]D cases further suggests that endog- enous vC]D has not occurred in this country. The median age at death of vC]D patients in the United Kingdom is 28 years (Andrews, 2003~. By con- trast, the median age at death among sC]D patients in the United States

28 30 25 20 - 15 ADVANCING PRION SCIENCE vCJD* (n = 87) .S. CJ D** (n = 1,358) 10 - 5- O- r I I I 1 1 1 1 1 1 1 r ~ \ \ \ 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 5-year age group *Data as of May 1, 2001. **Non-iatrogenic cases, 1995-1999. FIGURE 6-2 Percent distribution of vCTD cases in the United Kingdom and sCTD cases in the United States by age group at death, 1995-2001. Reprinted from Belay and Schonberger (2002) with permission from Elsevier. Copyright 2002 by Elsevier. between 1995 and 2001 was 68 years. Figure 6-2 illustrates the strikingly different age distributions of vC]D and sC]D cases (Belay and Schonberger, 20021. In fact, less than 0.2 percent of noniatrogenic C]D patients in the United States died before the age of 30 (Belay and Schonberger, 20021. Table 4-1 in Chapter 4 provides a more extensive comparison of vC]D and sC]D. The one known U.S. resident with probable vC]D, a 23-year-old British woman who had resided in Florida since 1992, was born and raised in the United Kingdom during the BSE outbreak and is believed to have contracted vC]D from the consumption of infected beef products in her native country (Belay et al., 2003; CD C, 20021. She was still alive as of January 2004 (personal communication, E. Belay, C D C,January 16, 20041. At least 144 cases of vC]D, including the Florida woman, had been identified globally as of April 2003. The vast majority of these cases occurred in the United King- dom, but a few were identified in continental Europe, Hong Kong, and Canada.3 3The single cases of vCJD in Hong Kong and Canada are not considered endogenous be- cause the infected individuals had lived in the United Kingdom for an extended period of time during the BSE epidemic before effective controls were in place. Therefore, it is believed that these people became infected with the BSE agent in the United Kingdom.

SURVEILLANCE FOR TSEs IN THE UNITED STATES 129 At present, four scientists conduct surveillance and epidemiology for TSE fatalities at CDC's Division of Viral and Rickettsial Diseases at the National Center for Infectious Diseases. In fiscal year 2003, $3.5 million was budgeted for the TSE group (personal communication, E. Belay, April 20021. In addition to salaries and research, these funds are intended to cover the group's overhead at CDC, operation of NPDPSC, and support for state-based TSE surveillance programs. The CDC team reviews passively reported cases of human TSE and analyzes national mortality data to determine the trends in human TSE among the U.S. population (Belay et al., 20011. Suspected deaths from TSE are referred to NPDPSC, which makes arrangements for autopsies, collects clinical data, performs neuropathological and neurohistochemical exami- nations on samples of brain tissue, and analyzes PrP and PRNP from brain and other tissues (see Box 6-11. CDC also monitors populations with a known risk of accidental infec- tion by priors, such as recipients of human growth hormone (Belay, 20031. CDC's epidemiologists have examined mortality records4 for evidence of 4The investigators searched for death certificates that listed as causes of death (1) hemo- philia A (International Classification of Diseases [ICD]-9 code 286.0), (2) hemophilia B (ICD- 9 code 286.1), thalassemia (ICD-9 code 282.4), and sickle-cell disease (ICD-9 code 282.6) (Holman et al., 1996).

130 ADVANCING PRION SCIENCE diseases associated with increased exposure to blood or blood products (Holman et al., 1996~. Although substantial research has shown that sC}D and other long-known forms of human TSE are not transmissible through blood transfusions (Dodd, 2002; Foster, 2000), it remains unknown whether vC}D is transmissible in this manner. A hallmark of vC}D at present is the youth of its victims; therefore, CDC investigates all suspected cases of human TSE among those younger than age 55. To date, no evidence of endogenous vC}D or of other new variants of C}D has been discovered. As alluded to above, concern arose that the infectious agent of CWD had crossed the species barrier into humans after six cases of neurodegen- erative disease were detected in the United States in the l990s among hunt- ers and relatives of hunters (Belay et al., 2001; CDC, 2003~. However, follow-up investigations by CDC and state and local health authorities found no causal link between any of the six cases and CWD. The first investigation concerned three people aged 28 to 30 who died of C}D between 1997 and 2000 and who regularly ate deer or elk meat (Belay et al., 2001~. CDC aimed to determine whether the patients might have contracted a TSE by eating venison infected with the agent of CWD. The investigators concluded that the association of the three C}D patients with venison consumption "was more likely coincidental than causal" (Be- lay et al., 2001:1677-1678). In August 2002, CDC launched a similar investigation involving three men who died of neurodegenerative illnesses between 1993 and 1999 and who jointly participated in wild game feasts in northern Wisconsin (CDC, 2003~. The investigation revealed that the meat eaten at the feasts did not come from CWD-endemic areas. In addition, only one of the men had died of a TSE, and it was a common form of sC}D with no link to CWD. Thus, the investigators concluded that this second set of three cases also provided no evidence of a causal link between CWD and human neurodegenerative disease. National Prion Disease Pathology Surveillance Center CDC founded NPDPSC in collaboration with the American Associa- tion of Neuropathologists in 1996 to establish a national neuropathology laboratory for the analysis of human prion diseases (Belay, 2003~. NPDPSC examines the brain tissue and other informative tissues from all available5 possible and probable cases of human TSE in the United States. These ex- 5Many cases of human TSE in the United States are not identified, reported, or autopsied. This issue is discussed later in the chapter.

SURVEILLANCE FOR TSEs IN THE UNITED STATES 131 aminations, outlined in Box 6-1' provide data for monitoring and charac- terizing the occurrence of human TSEs. In addition, NPDPSC stores the tissue samples it collects for research purposes. From 1997 through May 2003' NPDPSC received 1,095 referrals, of which 60.4 percent were definite cases of prion disease. Table 6-1 shows the number of cases diagnosed by the center annually for each type of hu- man prion disease. According to NPDPSC, these figures underrepresent the total number of human TSE cases that have occurred in the United States by about 50 percent. This conclusion is based on the following analysis. NPDPSC diagnosed 304 U.S. cases of human prion disease among the 5116 referrals it received during 2001 and 2002 (NPDPSC, 2003; personal communication, P. Gambetti, NPDPSC, July 12, 2003~. However, since an estimated 1.07 cases of prion disease per million U.S. population occurred annually between 1979 and 2000 (Belay, 2003), one would expect that about 597 cases oc- curred in the United States in 2001 and 2002, as shown in Table 6-2. There- fore, only 51 percent of the estimated number of actual U.S. human TSE cases were identified and referred to NPDPSC during 2001 and 2002. Several factors probably account for the low referral rate to NPDPSC. Some physicians may not recognize human TSEs as such. Some physicians or staff may inadequately document a suspected TSE death. Some individu- als who die of TSE may be misclassified as dying from other, more common neurodegenerative conditions. Finally, many diagnosed cases of human TSE are not autopsied. Increasing the U.S. Autopsy Rate for Human TSE Deaths The only way to diagnose a human TSE definitively is through a neuro- pathological examination. The estimated U.S. autopsy and referral rate of 51 percent for human TSEs prevents comprehensive surveillance for these diseases in this country. Unfortunately, the U.S. autopsy rate has been falling for more than 50 years (Hoyert, 2001). The general autopsy rate in the United States dropped from around 35 percent in the 1960s (Shojania et al., 2003) to 9.4 percent in 1994 (National Center for Health Statistics, 1996), the last year for which CDC tabulated autopsy data. By 2003, the national, nonforensic rate was estimated at 5 percent (Shojania et al., 2003~. In contrast, the 51 percent referral rate to NPDPSC is quite an achievement. Nevertheless, to improve U.S. surveillance for human TSEs, clinicians' 6The case of vCJD in 2002 is excluded.

SURVEILLANCE FOR TSEs IN THE UNITED STATES 131 aminations, outlined in Box 6-1' provide data for monitoring and charac- terizing the occurrence of human TSEs. In addition, NPDPSC stores the tissue samples it collects for research purposes. From 1997 through May 2003' NPDPSC received 1,095 referrals, of which 60.4 percent were definite cases of prion disease. Table 6-1 shows the number of cases diagnosed by the center annually for each type of hu- man prion disease. According to NPDPSC, these figures underrepresent the total number of human TSE cases that have occurred in the United States by about 50 percent. This conclusion is based on the following analysis. NPDPSC diagnosed 304 U.S. cases of human prion disease among the 5116 referrals it received during 2001 and 2002 (NPDPSC, 2003; personal communication, P. Gambetti, NPDPSC, July 12, 2003~. However, since an estimated 1.07 cases of prion disease per million U.S. population occurred annually between 1979 and 2000 (Belay, 2003), one would expect that about 597 cases oc- curred in the United States in 2001 and 2002, as shown in Table 6-2. There- fore, only 51 percent of the estimated number of actual U.S. human TSE cases were identified and referred to NPDPSC during 2001 and 2002. Several factors probably account for the low referral rate to NPDPSC. Some physicians may not recognize human TSEs as such. Some physicians or staff may inadequately document a suspected TSE death. Some individu- als who die of TSE may be misclassified as dying from other, more common neurodegenerative conditions. Finally, many diagnosed cases of human TSE are not autopsied. Increasing the U.S. Autopsy Rate for Human TSE Deaths The only way to diagnose a human TSE definitively is through a neuro- pathological examination. The estimated U.S. autopsy and referral rate of 51 percent for human TSEs prevents comprehensive surveillance for these diseases in this country. Unfortunately, the U.S. autopsy rate has been falling for more than 50 years (Hoyert, 2001). The general autopsy rate in the United States dropped from around 35 percent in the 1960s (Shojania et al., 2003) to 9.4 percent in 1994 (National Center for Health Statistics, 1996), the last year for which CDC tabulated autopsy data. By 2003, the national, nonforensic rate was estimated at 5 percent (Shojania et al., 2003~. In contrast, the 51 percent referral rate to NPDPSC is quite an achievement. Nevertheless, to improve U.S. surveillance for human TSEs, clinicians' 6The case of vCJD in 2002 is excluded.

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134 ADVANCING PRION SCIENCE index of suspicion for this diagnosis must increase. Physicians and public health officials must identify more cases of human TSEs, more of those cases must be autopsied, and the relevant tissues must be sent to NPDPSC for a definitive diagnosis. Moreover, to identify new phenotypes of human TSEs, more cases of neurodegenerative diseases in general must be autopsied. Doing so would help CDC monitor the U.S. population for the theoretical transmission of CWD to humans. As noted above, there is to date no evidence that CWD is transmissible to humans. Should such transmission occur, however, the human form of CWD might bear little clinical resemblance to known hu- man TSEs hence the need to autopsy as many atypical cases of neurodegenerative disease as possible. The scientific history of FFI illus- trates this point. Unlike any other known TSE, the clinical hallmark of FFI is, as its name suggests, inherited progressive insomnia. Scientists began to suspect that FFI was a TSE only after neuropathological examination of FFI deaths revealed spongiform neurodegeneration (Manetto et al., 19921. Fur- ther studies confirmed the presence of proteinase K (PK)-resistant PrP in the decedents' brain tissues and a consistent point mutation (at codon 178) in their PRNP (Medori et al., 19921. The factors that contribute to the low autopsy rate of human TSE cases in the United States may include a cultural resistance to autopsies and the reluctance of most pathologists to conduct brain autopsies on suspected TSE cases for fear that the pathologist or his/her tools will become contami- nated. CDC is studying these issues through its Emerging Infections Pro- grams in New York and California (Belay, 20031. Preliminary data from a survey of pathologists in California indicate that about 80 percent of re- spondents do not perform autopsies on suspected CAD decedents because of concerns about infection control (Belay, 20031. In an effort to increase the referral and autopsy rates for suspected cases of human TSEs, NPDPSC began in December 2001 to cover the cost of an autopsy for a suspected CJD case; to identify institutions around the country willing to conduct reimbursed autopsies on suspected CAD cases; and, if necessary, to arrange and pay for the body to be moved to the closest location where an autopsy can be performed. If researchers found a way to inactivate prions on metal surfaces or remove prions from those surfaces, more pathologists probably would be willing to autopsy suspected TSE cases (see Chapter 71. In addition, the United States should evaluate and support ways to in- fluence neurologists, pathologists, and families to pursue autopsies of all cases of neurodegenerative disease, including suspected TSEs. One solution could be to communicate more frequently with pathologists and neurolo- gists to remind them of both the need to autopsy suspected TSE patients and the free services available for doing so. After the director of NPDPSC

SURVEILLANCE FOR TSEs IN THE UNITED STATES 135 made a presentation about the TSE surveillance program at a national pa- thology meeting in 2000, the number of referrals to the center increased significantly. As of Tuly 2003, NPDPSC had approached five national asso- ciations of pathologists and neuropathologists in an effort to encourage their members to submit cases of suspected human TSEs to the center. Also, NPDPSC and CDC are seeking support from state health departments to urge American neurologists to report cases of suspected human TSEs (per- sonal communication, P. Gambetti, NPDPSC, April and July, 20031. Recommendation 6.1: Provide funds to promote an increase in the proportion of cases of human neurodegenerative disease, especially suspected cases of transmissible spongiform encephalopathy, that are recognized and autopsied. [Priority 217 The Need for More Epidemiological Research to Improve TSE Surveillance The committee sees a number of opportunities for the United States to improve its ability to detect a new human TSE, including a theoretical case caused by the infectious agent of CWD. Although CDC and NPDPSC have conducted many valuable laboratory and epidemiological investigations and analyses, the comparison in Table 6-3 of the United States' CJD surveil- lance program with those of the United Kingdom and Canada illustrates the relatively limited amount of resources that this country devotes to CTD surveillance and epidemiology. The improvement of U.S. surveillance for human TSEs will depend largely on information gleaned from epidemiological studies that help de- fine the target population and hone survey instruments. The 3 to 4 percent of autopsied cases that fall outside the classified subtypes of human TSEs could be studied further to determine whether a new human TSE lies among these atypical cases (personal communication, P. Gambetti, NPDPSC, March 26, 20031. Risk factors for CTD could be identified by studies that examined differences between CTD cases and controls. Comparing the age and geographic distribution of all neurodegenerative diseases in the United States with the distribution of all CTD cases might suggest regions in which CTD is underreported. In addition, the validity of death certification for CJD could be examined by comparing data on death certificates with re- sults of neuropathological exams. Death certificates also could be examined to determine whether significant differences exist between the demograph- 7The committee denotes each recommendation as priority level 1, 2, or 3 based on the criteria and process described in the Introduction.

136 ADVANCING PRION SCIENCE TABLE 6-3 Comparison of National Surveillance and Epidemiology Programs for Human TSEs in the United States with Those in the United Kingdom and Canada Characteristic of the Program United States Canada United Kingdom Funding ($U.S.; FY2003)a $3.5 million $0.55 million $3.2 million Funding per capitab 1.2 cents 1.7 cents 5.4 cents Average annual number of 256 91.3 167.5 referrals of suspect human TSE casesC ~ Average annual number of 0.90 2.94 2.86 referrals of suspect human TSE cases per 1 million populatione Stafif 13 14 full-time 26 full-time equivalents equivalents Is CJD a nationally No No No reportable disease? aFY = fiscal year. U.S. data: personal communication, E. Belay, CDC, April 21, 2003; the U.S. 2003 fiscal year is October 1, 2002-September 30, 2003. Canadian data: personal communication, N. Cashman, Health Canada, August 26, 2003; the Canadian 2003 fiscal year is April 1, 2003-March 31, 2004. Currency converted to $U.S. using the interbank conversion rate on April 1, 2003 of $1 U.S. = $1.468 Canadian (FXConverter, 2003). United Kingdom data: personal communication, A. Hunter, National Creutzieldt-Jakob Disease Surveillance Unit, July 21, 2003; the U.K. 2003 fiscal year is April 1, 2003-March 31, 2004. Currency converted to $U.S. using the interbank conversion rate on April 1, 2003 of $1 = £0.63 (FXConverter, 2003). bProjected resident population of the United States in 2003 = 282.8 million (U.S. Census Bureau, 2000). Projected population of Canada in 2003 = 31.5 million (Statistics Canada, Demography Division, 2003). Projected population of the United Kingdom in 2003 = 59.2 million (Government Actuary's Department, 2002). The number of years included in the average varies by country and excludes each surveillance system's start-up years. For the United States: 2001-2002; Canada: 2000-2002; United Kingdom: 1997-2002. ~Sources: for the United States: NPDPSC (2003); Canada: Health Canada (2003); United Kingdom: National Creutzieldt-Jakob Disease Surveillance Unit (2003). eEstimated average annual civilian resident population of the United States, 2001-2002 = 285.6 million (U.S. Census Bureau, 2002, 2003). Estimated average annual population of Canada, 2000-2002 = 31.105 million (Statistics Canada, 2003). Approximate average annual population of the United Kingdom, 1997-2002 = 58.6 million (Office of National Statistics, 2003). Sources: United Kingdom: personal communication, A. Hunter, National Creutzieldt- Jakob Disease Surveillance Unit, July 21, 2003; United States: personal communication, P. Gambetti, NPDPSC, 2003; Canada: personal communication, N. Cashman, Health Canada, August 26, 2003.

SURVEILLANCE FOR TSEs IN THE UNITED STATES 137 ics of autopsied and nonautopsied CAD cases, as well as what factors are . . . . . . . . . . . associates Wlt" ~ participation or nonparticipation in autopsies, a question on which CDC has launched pilot studies in two states. Other investiga- tions could be conducted to characterize the frequency and types of human exposure to CWD in the United States. For example, studies could be done comparing the mortality rates of human TSEs over time in CWD-endemic areas with the rates in non-CWD-endemic areas. The Colorado Depart- ment of Public Health and Environment is presently reviewing the death certificates of state residents from the past 32 years to compare the rates of death due to a range of neurological diseases, including CAD, in CWD- endemic and nonendemic regions of the state (Quirk, 20021. More such studies should now be initiated in other CWD-endemic states. These are just a few of the many epidemiological studies that would provide a firmer scientific foundation for surveillance of human TSEs in the United States. Recommendation 6.2: Provide funds to increase the number and diversity of epidemiological studies on human transmissible spongiform encephalopathies (TSEs) in the United States. In par- ticular, support research to identify potential cases of variant Creutzfel~t-lakob disease and new human TSEs possibly caused by the agent of chronic wasting disease. [Priority 21 U.S. SURVEILLANCE FOR TSEs IN ANIMALS The three major TSEs of animals scrapie, BSE, and CWD pose vary- ing degrees of risk to animal and human health in the United States. First, no scientific evidence has emerged during the past two and a half centuries that scrapie of sheep and goats causes a TSE in humans. However, scrapie spreads easily among sheep and goats, is transmissible to ruminants through animal feed, could possibly be transmissible to cervids, and exists in the United States. Second, the infectious agent of BSE is widely believed to cause vCJD, but, as noted earlier, there has been no known case of BSE in the United States. Considerable epidemiological evidence suggests that the disease does not spread horizontally (from an infected live cow or bull to a healthy one) or vertically (from an infected cow to her fetus). Finally, existing evidence neither confirms nor rules out the possibility that CWD could infect humans. Scientists in the laboratory of Dr. Stanley Prusiner attempted to transmit CWD to transgenic mice that express hu- man PrP, but the mice remained healthy (personal communication, M. Sear, Institute for Neurodegenerative Diseases, Tune 20, 20031. Epidemiologists have not identified a causal link between a case of human neurodegenerative disease and CWD after investigations into six suspect cases (Belay et al., 2001; CDC, 20031. Yet two lines of evidence suggest that humans could

138 ADVANCING PRION SCIENCE conceivably develop a TSE by eating venison infected with PrPCwD. First, investigators from the National Institutes of Health (NIH) induced the con- version of human prpC to PrPCwD in a cell-free medium (Raymond et al., 2000~. Second, as described above, BSE has crossed the species barrier from cattle to humans and has caused vCTD. Finally, scientists have predicted that CWD could potentially wipe out portions of the U.S. wild deer popula- tion if the epidemic is not curtailed (Gross and Miller, 2001~. These concerns justify robust surveillance for animal TSEs in the United States. The following sections describe the U.S. surveillance programs for these three diseases and present the committee's recommendations for re- search to strengthen those programs. Surveillance of Ovines for Scrapie Scrapie, a TSE of sheep and goats, was first described in the 1700s (Godon and Honstead, 1998~. The first U.S. case was identified in 1952 in an animal in Michigan that was imported from the United Kingdom through Canada (personal communication, L. Detwiler, independent consultant, August 27, 2003~. Although all routes of transmission are not known, it has been shown that the scrapie agent can be transmitted laterally (from one unrelated animal to another), maternally (from an infected ewe to her off- spring during pregnancy or lambing), and iatrogenically (Detwiler and Baylis, 2003~. Clinical signs of the disease appear 2 to 4 years after infec- tion, and affected animals progress invariably to death. Epidemiological studies, limited primate inoculation studies, and in vitro molecular studies all have indicated that the infectious agent of scrapie is not transmissible to humans, as noted above. Some U.S. cattle that were experimentally inoculated by various routes with the U.S. strain of the scrapie agent appear to have developed an encephalopathy that is not BSE (Clark et al., 1995; Cutlip et al., 1994, 1997~. Scrapie has posed a financial risk to the owners of the United States' approximately 6.35 million sheep and 1.2 million goats (NASS, 2003~. Not only does the disease reduce the productivity of infected flocks, but it also prevents the export of breeding stock, semen, and embryos to many other countries (Veterinary Services, 2002~. Figure 6-3 depicts the reported occurance of scrapie during fiscal year 2002. Until recently, the estimated prevalence of scrapie in U.S. sheep was 0.07 percent (personal communica- tion, N. E. Wineland, USDA APHIS, September 8,2003~. USDA's surveillance program for scrapie, which includes goats but fo- cuses on sheep, depends partially on flock owners' participation in a certifi- cation program administered by Veterinary Services of USDA's Animal and Plant Health Inspection Agency (APHIS) (Williams, 2002~. Approximately 2 percent of the country's farmed sheep, or about 130,000 animals in 1,755

SURVEILLANCE FOR TSEs IN THE UNITED STATES `; ~ 2 ~,3 2 139 11 1 54 i ~` Total = 379 VIA 21 1 1 ~ 1 g11 / = 10 i\ 7 69 1 ~1 , F~8 \ ~ 1 ~ ~ 2 20 ~ 14~ ~ 7,'; 2 DO \~ ~ ~ ~ FIGURE 6-3 Locations of 375 of 379 confirmed cases of scrapie reported to USDA APHIS's Veterinary Services during the 2002 fiscal year. The level of scrapie surveillance varies greatly among the states. NOTE: Four validation cases confirmed in FY 2002 are not shown on map. SOURCES: personal communcation, E. Williams, University of Wyoming, July ll, 2003; Veterinary Services, USDA APHIS (2003a). flocks, were registered in the certification program as of Tuly 2003 (NASS, 2003; personal communication, L. Detwiler, independent consultant, Au- gust 27, 2003; Veterinary Services, 20031. There were 64,170 U.S. sheep operations in 2002 (NASS, 20031. Figure 6-3 illustrates the reported occur- rence of scrapie by state during fiscal year 2002. Given the small percentage of sheep monitored through the certifica- tion program, Veterinary Services launched a year-long study in April 2002 to determine the prevalence of scrapie through the collection and analysis of tissue samples from mature sheep at slaughterhouses (personal commu- nication, L. Detwiler, independent consultant, August 27, 20031. In fact, about 60 percent of sheep tissue samples received by the National Veteri- nary Services Laboratories (NVSL) during fiscal year 2002 came from the culled slaughterhouse population (Veterinary Services, 2003a). Although the data from the slaughter surveillance study were still under analysis at the time of this writing, it appeared that the results would yield a revised estimate of the prevalence of scrapie in this country (personal communica- tion, N. E. Wineland, Veterinary Services, September 8, 20031.

140 ADVANCING PRION SCIENCE Veterinary Services also is conducting a small amount of targeted, ac- tive surveillance to validate a new antemortem diagnostic test for scrapie and gather data for regulatory approval of the test (O'Rourke et al., 2002; Veterinary Services, 2003a). A mandatory traceback study begun in April 2003 aims to trace infected animals identified during slaughterhouse sur- veillance back to their source herds (personal communication, L. Detwiler, independent consultant, August 27, 20031. Despite these varied sources of data, however, more surveillance is required to accurately estimate the prevalence of scrapie in the United States (personal communication, E. Wil- liams, University of Wyoming, May 20031. Goals of Scrapie S?~rveil~nce USDA conducts scrapie surveillance as part of a program begun in the mid-19OOs to eradicate the disease from the United States. The eradication program has not succeeded in the United States or in most other countries where scrapie has become endemic (Detwiler and Baylis, 20031; however, Iceland appears close to wiping out the disease (personal communication, L. Detwiler, independent consultant, August 27,20031. U.S. efforts to eradi- cate scrapie have intensified since the scientific community reached the pre- vailing consensus that scrapie-infected sheep offal in cattle feed sparked the BSE epidemic in the United Kingdom (Wilesmith et al., 19881. Through surveillance, USDA aims to prevent scrapie-infected sheep from infecting other animals in their flock, from being sold to owners of a healthy flock, and from contaminating pastures with the infectious agent of the disease. The tissue samples collected during scrapie surveillance also have been used for the validation of an antemortem test that employs immunohistochemis- try to detect prions in a tissue sample from a sheep's nictitating membrane (third eyelid)8 (O'Rourke et al., 2002), as well as in research projects aimed at developing better techniques for scrapie management and control (per- sonal communication, E. Williams, University of Wyoming, May 20031. Scrapie Flock Certification Program The Scrapie Flock Certification Program, begun in 1992, helps flock owners identify scrapie-free flocks from which to purchase new animals (Veterinary Services, 19981. About 3 percent of U.S. sheep operations were 8Prions have been found in the third eyelids of asymptomatic sheep infected with scrapie (O'Rourke et al., 2000) but not in the third eyelids of cattle or cervids infected with a TSE. This difference is due to the differing paths that prions travel on their way to the central nervous system in different animals.

SURVEILLANCE FOR TSEs IN THE UNITED STATES 141 registered in the program as of Tuly 2003. As mentioned earlier, this corre- sponds to about 2 percent of farmed sheep. Participating flocks are monitored for 5 years or more. Owners must record all acquisitions, departures, births, and deaths of their sheep, and must keep these records for a minimum of 5 years after an animal dies or is removed from the flock. These records aid in epidemiological efforts to trace the source of scrapie infections. If no animal in a monitored flock is diagnosed with scrapie during the 5-year period and none shows clinical signs of the disease, the flock is certified as scrapie-free. Since sheep from a certified flock fetch a higher price than other sheep, the market creates an . . . . . . economic Incentive to participate In t" He program. If NVSL diagnoses a case of scrapie, officials conduct an epidemiologi- cal investigation to determine the source of the infection and identify sheep that were exposed to the infected animal. Veterinary Services works with the state and the flock owner to develop a scrapie eradication plan. Those sheep determined to be at high risk of contracting the disease are culled from the flock, and Veterinary Services helps the flock's owner clean and disinfects the sheep facilities. USDA epidemiologists also conduct traceback studies to determine the source of scrapie infections discovered in animals at slaughter. As increas- ingly more sheep are registered in USDA's individual animal identification program, epidemiologists will have a better too! for tracking scrapie-in- fected sheep back through the herds in which they lived (personal commu- nication, E. Williams, University of Wyoming, July 11, 20031. As men- tioned earlier, 60 percent of the tissue samples examined for scrapie came from slaughterhouses in fiscal year 2002, but this represents a small amount of slaughterhouse surveillance relative to what Veterinary Services plans to do (personal communication, E. Williams, University of Wyoming, May 2003). Statistics on Scrapie Infection NVSL confirmed 193 cases of scrapie between October 1, 2002, and March 31, 2003, and identified 27 flocks that contained one or more cases of scrapie or were the source of a scrapie-infected animal (Veterinary Ser- vices, 2003b). A cumulative total of 49 flocks contained scrapie-infected sheep or were the source of an infected animal as of March 31, 2003. As noted earlier, however, these statistics do not reflect the true prevalence of 9As discussed in Chapter 7, disinfecting anything that has had contact with an agent of TSE from a pasture to a scalpel is extremely difficult. In most cases, scientists do not yet . . . . . . . . know . tow to e. Emanate proton ~ntect~v~ty.

42 ADVANCING PRION SCIENCE scrapie in the United States because a limited number of flock owners par- . . . ... . t~c~pate In t" be cert~cat~on program. Surveillance of Bovines for BSE As discussed above, no evidence of BSE has been detected in the United States. The disease became reportable in 1987, meaning that since then, USDA has required notification of all BSE cases (Brown et al., 2001). USDA began to conduct active surveillance for BSE in 1990. At present, the surveillance program targets the subpopulations of the 45 million adult cattle in the United States that would be most likely to have BSE (Williams, 2002; PL 107-9 Federal Inter-agency Working Group, 20031. These subpopulations include cattle on farms that exhibit signs of neurological disease, cattle condemned at slaughter for neurological dis- ease, cattle with neurological disease that are referred to veterinary diag- nostic laboratories, rabies-negative cattle submitted to public health labora- tories, cattle that are unable to rise from a recumbent position (often called downer cattle or fallen stock), and cattle that die of unknown causes on farms (Williams, 2002; PL 107-9 Federal Inter-agency Working Group, 2003; USDA APHIS, 2003a). In 2002 USDA significantly increased the pro- portion of tissue samples obtained for BSE testing from this last subpopula- tion (USDA APHIS, 2003b). In addition to the above measures, veterinary pathologists at zoos in the United States conduct neuropathological examinations of animals that exhibited signs of neurological disease before death (USDA APHIS, 2003a). They do so because the United Kingdom's early cases of BSE included some zoo animals (Jeffrey and Wells, 1988; Kirkwood and Cunningham, 19941. Until 1993, the USDA surveillance program for BSE used exclusively histopathological examinations of brain tissue to search for physical evi- dence of spongiform encephalopathy. In 1993, the program adopted immu- nohistochemistry and the Western blot to determine whether prions were present in tissue samples (Detwiler, 20031. Today, all the program's neuro- pathological examinations rely on immunohistochemistry, and some are performed in conjunction with histopathology and Western blot (Detwiler, 20031. Historically, USDA and its collaborators have sampled cattle-brain tis- sues for BSE at approximately double the rate recommended by the Office International des Epizooties (OIE), which sets the worId's standards for animal health (PL 107-9 Federal Inter-agency Working Group, 20031. The sampling rate rose to 5 times the OIE standard in 2000 and to more than 10 times the standard in 2001 (PL 107-9 Federal Inter-agency Working Group, 20031. As shown in Figure 6-4, tissue samples from 19,900 cattle were tested

SURVEILLANCE FOR TSEs IN THE UNITED STATES 25,000 20,000 In In .= 1 5,000 Q - i~ C' ~ 0 000 Q z 5,000 o 143 I I T T I I T I I I T '91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02 Year FIGURE 6-4 Number of cattle brains tested for BSE per year in the United States. SOURCE: USDA APHIS (2002). for BSE in 2002 (USDA APHIS, 2002), surpassing the goal of 12,500 samples set for the 2002 fiscal year. Testing at least 12,500 brain-tissue samples would theoretically permit the detection of one or more clinical cases per million adult cattle, given the estimated number of adult cattle in the U.S. subpopulations most vulnerable to BSE. USDA decided to aim for the level of one per million because classic C}D appears to occur clinically at the rate of one case per million population, and because TSE researchers have hypothesized that other spongiform encephalopathies may also occur at that rate in any given population (Brown et al., 2001; USDA APHIS, 2002~. This rationale may be inappropriate for the detection of endemic, foodborne cases of BSE, however, because such cases would occur at a higher prevalence than spontaneous TSEs in cattle. USDA APHIS locates, monitors, and offers to purchase cattle that were imported to the United States from countries now known to have BSE or to be at high risk of a BSE outbreak, including the United Kingdom, Ire- land, countries in continental Europe, and Japan (USDA APHIS, 2003a;

44 ADVANCING PRION SCIENCE PL 107-9 Federal Inter-agency Working Group, 2003~. The purchased cattle are sacrificed and tested for BSE. As of this writing, all of these animals have tested negative for BSE (PL 107-9 Federal Inter-agency Work- ing Group, 2003~. As of March 2003, USDA APHIS was aware of and monitoring seven live cattle that had been imported from BSE-reporting countries before the practice was banned. Between 1991 and 2001, three studies assessing the risk of BSE in the United States led to the conclusion that the country is highly resistant to the introduction and spread of the disease (Health and Consumer Protection Directorate-General, 2000; HCRA and TUCCE, 2001; Veterinary Services, 1991a,b). The most recent study is discussed in detail in Chapter 7, but we address its surveillance aspects here. The 2001 risk assessment, commissioned by USDA, underscored the importance of surveillance for managing the risk of BSE (HCRA and TUCCE, 2001; PL 107-9 Federal Inter-agency Working Group, 2003~. The authors state that an important source of uncertainty in their mode! was the proportion of clinical cases of BSE that are correctly identified during ante- mortem surveillance at slaughter (HCRA and TUCCE, 2001:99~. The au- thors' best- and worst-case assumptions for this proportion ranged from 99 percent to 50 percent, with a base level of 90 percent. Fewer than 4 months after the release of the Harvard/Tuskegee study, the U.S. General Accounting Office (GAO) issued a report, requested by Sens. Tom Harkin, Richard G. Lugar, and Richard }. Durbin, critiquing aspects of the U.S. government's risk-management program for BSE (GAO, 2002~. One criticism of the program was that the target population for BSE surveillance included relatively few cattle that died on farms. In unpub- lished comments addressed to GAO on its draft of this report, USDA re- sponded that, very recently, it had begun to test cattle that died on farms of unknown causes. Plans were in place to increase the proportion of these cattle tested in fiscal 2002, the agency added.~° USDA reiterated this point in its published response to GAO's recommendations (GAO, 2002:56; USDA, 2002~. Surveillance of Cervids for CWD The nationwide prevalence of CWD is unknown at present as a result of inadequate surveillance. However, the participants in a federally man- dated effort begun in May 2002 aim to change that situation (CWD Task Unpublished document provided to the committee by L. Detwiler of USDA APHIS in March 2003.

SURVEILLANCE FOR TSEs IN THE UNITED STATES 145 Force, 2002~. Indeed, a few states, including South Dakota, Colorado, and Wyoming, have already established highly effective CWD surveillance and eradication programs. Yet a uniform national surveillance program for CWD has been difficult to implement in the United States because most states, federal agencies, and the U.S. Congress did not perceive the disease to be a national problem, and because the regulation of cervids is largely the province ot states. The alarming discovery of CWD in white-tailed deer shot by hunters in Wisconsin in autumn 2001 prompted Congress to instruct USDA and the Department of the Interior (DOI) to establish a national CWD management program (holy et al., 2003; USDA and DOI,2002~. The two agencies jointly established a multijurisdictional, multisector CWD Task Forceii that they cochair (CWD Task Force, 2002~. The discovery of CWD in Wisconsin deer also prompted Congress to give USDA APHIS line-item funding for CWD for the first time. The fiscal year 2003 budget allocated almost $15 million to APHIS: $7.2 for farmed cervids and $7.7 for free-ranging cervids (personal communication, D. E. Goeldner, USDA APHIS, May 2, 2003~. The CWD Task Force used that money to begin to implement its plan, which includes comprehensive nationwide surveillance for the disease (USDA and DOI, 2002~. The funds are sorely needed, as many states have to date lacked the resources and infrastructure necessary to conduct CWD surveillance. Congress has allocated more funds to APHIS for CWD sur- veillance in the 2004 fiscal year (personal communication, Lynn Creekmore, USDA APHIS, October 10, 2003~. The committee supports the plan out- lined by the task force's surveillance working group, discussed later in this chapter. The congressional allocation also is being used to support epide- miological studies, the testing of thousands of cervid tissue samples from the 2002-2003 hunting season, the evaluation of new rapid-test diagnos- tics, and the investigation of a vaccine for CWD, among other activities (personal communication, D. E. Goeldner, USDA APHIS, May 2, 2003~. Spread of CWD Across the United States At least 5.5 million free-ranging mule deer, 14 million free-ranging white-tailed deer, and 1 million free-ranging elk reside in the United States (Nowak, l999~. In addition, 130,000 to 200,000 elk and an unknown num- iiThe CWD Task Force includes USDA, DOI, 14 state agencies concerned with wildlife management and agriculture, four universities, the Southeastern Cooperative Wildlife Disease Study, and the International Association of Fish and Wildlife Agencies. The task force was formed in response to a congressional directive that USDA and DOI assist state wildlife man- agement and agriculture agencies with the management of CWD (CWD Task Force, 2002).

146 0~ ADVANCING PRION SCIENCE / ~ '~l ~ f / - at- = FIGURE 6-5 North American locations where CWD had been diagnosed as of May 2003. The darkest areas represent locations where CWD has been found in free- ranging cervids. The grey areas represent states and provinces where CWD has been diagnosed in captive cervids. Some of these CWD-positive captive herds have been depopulated. The grey circle near Toronto represents a location where CWD occurred in the 1970s and where the disease has not persisted. SOURCE: personal communication, E. Williams, University of Wyoming, May 2003. ber of deer live on American game farms, primarily in western, midwestern, and northeastern states (Williams, 20021. CWD was first recognized as a syndrome of cervids at a Colorado research facility in the 1960s (Williams and Young, 19801. It was first observed in free-ranging U.S. cervids in the 1980s (Spraker et al., 1997) and in farmed U.S. cervids in 1997 (Creekmore, 20021. The disease probably arose at least 40 years ago (Miller et al., 20001. Between 1996 and 2002, CWD apparentlyi2 spread from the core en- demic region illustrated in Figure 6-5 a contiguous area of northeastern i2~t iS also possible that increased awareness of CWD has brought preexisting cases to light for the first time.

SURVEILLANCE FOR TSEs IN THE UNITED STATES 147 Colorado and southeastern Wyoming to captive and free-ranging herds in Illinois, Kansas, Minnesota, Montana, Nebraska, New Mexico, Oklahoma, South Dakota, Wisconsin, Utah, and the Canadian provinces of Saskatchewan and Alberta (Williams and Miller, 2002; Fischer and Nettles, 2003~. Reports of the disease in states east of the Mississippi were particu- larly alarming because CWD previously had been considered a western problem alone (CWD Task Force, 2002~. Figure 6-5 also illustrates the locations of the farmed and free-ranging cervids in North America known to be infected with the agent of CWD. Helping to Prevent the Spread of CWD Through Surveillance Scientists who study CWD expect the disease to continue spreading for the foreseeable future and to be found in new locations as surveillance im- proves. The results of fiscal year 2003 surveillance expanded the known distribution of CWD infectivity among wild cervids in such states as Wyo- ming and Colorado; however, the disease was not found in wild cervids outside of the eight states where it had been previously identified (personal communication, Lynn Creekmore, USDA APHIS, October 10, 2003~. In addition to the theoretical risk posed by CWD for humans who con- sume venison, the disease threatens the economic viability of the farmed cervid industry and could significantly impact hunting and its associated economies. Finally, models of CWD dynamics suggest that the epidemic could drive deer populations to extinction if infected herds are not identi- fied and culled while the prevalence of the disease in the herd is low (Gross and Miller, 2001~. Many factors may be contributing to the expanding epidemic. They include the ease of lateral transmission of the infectious agent of CWD; the movement of cervids along natural migration corridors; the unknown mechanism of transmission; environmental contamination by the agent lead- ing to the infection of more cervids (particularly at game farms where sick herds have been eradicated); the lack of efficient live-animal diagnostic tests; the lack of treatments for CWD; and the lack of inactivation techniques for the infectious agent (Gross and Miller, 2001; Williams and Miller, 2002~. Well-planned and -implemented CWD surveillance programs can en- able states and other organizations to detect new outbreaks of the disease early and to take actions to prevent its spread. A nationally integrated pro- gram of this sort would enable a central organization to efficiently monitor the prevalence, incidence, and distribution of CWD in the United States, to identify national trends in the disease, to support epidemiological studies of CWD, and to evaluate intervention strategies. The fact that each state has jurisdiction over its own deer and elk populations has complicated nation- wide data collection on the disease (personal communication, E. Williams,

148 ADVANCING PRION SCIENCE University of Wyoming, May 2003). For these reasons, the committee sup- ports the development of a comprehensive, unified surveillance program for CWD in cervids. Recommendation 6.3: Provide funds to support the development of a nationwide surveillance system for chronic wasting disease in the United States. [Priority 21 Present CWD Surveillance in the United States Some elements of a national CWD surveillance system are already evolv- ing. USDA APHIS has conducted surveillance for CWD in free-ranging cervids since 1997 in cooperation with a number of state wildlife and agri- culture agencies. During the 2002-2003 hunting season, program partici- pants collected more than 126,000 tissue samples from 37 states more than 10 times the number of samples tested during the 2001-2002 hunting season but primarily from two states: Colorado and Wyoming (personal communication, L. Detwiler, USDA APHIS, April 28, 2003~. All 50 states have accepted an offer from USDA APHIS for fiscal year 2004 funds to conduct CWD surveillance and related activities of the states' design (per- sonal communication, Lynn Creekmore, USDA APHIS, October 10, 2003~. In addition, the National Park Service conducts targeted surveillance of cervids exhibiting clinical signs of CWD and active surveillance of deer in parks located near confirmed CWD cases (CWD Task Force, 2002~. The extent of CWD surveillance in farmed cervids ranges from manda- tory monitoring and certification to no surveillance at all, depending on the state (CWD Task Force, 2002~. Since September 2001, APHIS has con- ducted surveillance of farmed elk herds known to harbor CWD-infected animals (USDA, 2001~. As of June 2002, 23 states were testing captive cervids for CWD, and 12 more states were in the process of developing farmed-cervid surveillance programs (CWD Task Force, 2002~. More than 6,200 tissue samples from farmed deer and elk were tested for CWD in fiscal year 2002 (Creekmore, 2002~. Goals for Improving CWD Surveillance The surveillance working group of the CWD Task Force estimates that the United States must test 150,000 cervids for CWD per year to monitor the disease adequately (CWD Task Force, 2002~. The country reached at least 84 percent of that goal in 2003, based on the aforementioned number of tissue samples collected. To guide all states toward the level of surveil- lance needed to adequately monitor CWD, the surveillance working group established the four broad goals and action items shown in Box 6-2. The

SURVEILLANCE FOR TSEs IN THE UNITED STATES 149 working group aims to foster the development of techniques for enabling each state to share the results of its unique CWD surveillance program such that the combined data could be analyzed on the national level (personal communication, Lynn Creekmore, USDA APHIS, October 10, 20031. In addition, the working group proposed a national program to certify herds of farmed cervids as being CWD-free (CWD Task Force, 20021. This pro- gram would require owners to report all cervid deaths and to have CWD testing performed on all animals (except calves) that are slaughtered or die on the premises. The committee supports the aims of the CWD Task Force's surveillance working group. We encourage the National Prion Research Program (NPRP) to fund projects that complement the group's activities. TSE Surveillance as an Antidote to Terrorism Adequate surveillance for TSEs is essential to discount and discredit terrorist hoaxes of planting BSE (WorId Health Organization, 20021. Re- search that helps produce better TSE diagnostics and better screening tests for infectivity would improve the United States' capability to conduct TSE

150 ADVANCING PRION SCIENCE surveillance and provide convincing reassurance to the public, thereby un- dermining TSE agents as a tool for terrorism. The committee determined that TSE agents would be ineffective tools for terrorism if the objective were to create an epidemic of TSE. The diffi- culty of artificially infecting animals or people with the agents, the ineffi- cient transmission of the agents within and between species (except cervids), and the long incubation period of TSEs make TSE agents unlikely weapons. However, the lack of reliable diagnostic tests for TSEs and the limited un- derstanding of the epidemiology and pathogenesis of these diseases could provide the conditions for an effective terrorist hoax (National Research Council, 20021. For example, if either a BSE hoax or a real case of BSE were effectively planted, it could damage consumer confidence in beef products, resulting in significant losses to the beef and cattle industries. ESSENTIAL RESEARCH TO IMPROVE U.S. CAPABILITIES TO CONDUCT SURVEILLANCE FOR TSEs Many unanswered questions regarding the epidemiology, natural his- tory, and prevalence of animal and human TSEs in the United States ham- per the nation's ability to conduct the best possible surveillance for these diseases. The limitations of current diagnostic tools for TSEs also pose ob- stacles to comprehensive surveillance. The committee recommends that NPRP support the research outlined in Box 6-3 because the results of such research would not only improve the quality of TSE surveillance in the United States, but also would advance prion science in general. These re- search recommendations are discussed in detail in the following sections. Transmission Characteristics Although not a single case of C W D has been diagnosed in humans, BSE has crossed the human species barrier. Therefore, it is important to deter- mine whether the theoretical risk of C W D transmission to humans is real. Making this determination will require a substantial amount of experimen- tal and epidemiological research, although work along these lines has be- gun. For example, studies in a cell-free conversion model have shown that the infectious agent of C W D can convert human prion protein (Caughey, 2001~. New animal models, such as the ferret (Sigurdson et al., 2003) and mice with C W D transgenes, are being developed for use in C W D transmis- sion experiments, as are cynomologous macaques (Lasmezas et al., 2001~. Nonhuman primates are particularly valuable surrogates for assessing the risk of transmission of the C W D agent to humans. The utility of cynomologous macaques as human surrogates in TSE transmission studies was demonstrated through experiments that further established BSE as the

SURVEILLANCE FOR TSEs IN THE UNITED STATES 151 cause of vC]D (Lasmezas, 20031. Lasmezas and colleagues showed that both the BSE and vC]D agents were readily transmitted to macaques by either the intracerebral (i.c.) or intravenous (i.v.) route, and that the trans- mission of either agent by either route resulted in the same histopathologi- cal lesions in the macaques' brains. Further, the scientists observed identical histopathological lesions in second-order macaques that were infected i.c. Or i.v. with either the vC]D or BSE agent from the previously infected macaques. These observations supported the hypothesis that the same prion strain causes both BSE and vC]D (Lasmezas et al., 20011. Moreover, Lasmezas and colleagues observed prions in the tonsils, spleens, and lymph nodes of the macaques observations also made in hu- mans with vC]D (Lasmezas, 2003 ). This finding suggests that cynomologous macaques would be excellent models for evaluating the risk

152 ADVANCING PRION SCIENCE of iatrogenic transmission of vCTD to humans and the transmissibility of the CWD agent to humans by various routes. Studies are needed to determine the transmissibility of CWD not only to humans, but also to other animal species. Several investigations are un- der way to determine whether the CWD agent is transmissible to cattle. In one such study, 5 of 13 cattle inoculated i.c. with CWD-infected tissue from the same mule deer developed a disease resembling TSE (Hamir et al., 2001; Hamir and Miller, 2002; USDA and FDA, 20031. In another ongoing study, cattle exposed in 1997 to the CWD agent by either oral inoculation or contact with CWD-affected cervids had shown no evidence of transmission as of Tuly 2003 (personal communication, E. Williams, University of Wyo- ming, Tuly 11, 20031. Similar studies should be performed with other do- mestic and wild animals using various routes of transmission, including the oral route. For example, CWD transmission studies in cervids' feline preda- tors, such as cougars, would be valuable. Exposure Characteristics The natural routes and vehicles of exposure to TSE agents and the mechanisms by which the agents invade a host are well defined for some TSEs and poorly defined for others. Considerable epidemiological and ex- perimental evidence underscores the importance of the oral route, inges- tion, and alimentary uptake of the agents of scrapie in sheep, BSE in cattle, vCTD in humans, and transmissible mink encephalopathy in mink. Yet little is known about the routes and vehicles by which cervids are exposed to the CWD agent. Nor is it known whether the agent of CWD can infect pri- mates (as a proxy for humans) after oral or cutaneous exposure to CWD- infected cervid tissues. Investigations into the route or routes by which healthy cervids become exposed to CWD and into the mechanisms of CWD infection are under way at a few U.S. laboratories and field sites. The committee urges NPRP to support these long-term investigations and fund complementary ones. For instance, experiments should be performed to determine whether cynomologous macaques can become infected with the CWD agent through oral or cutaneous exposure. . . . . . . . Host Susceptibility Susceptibility to a TSE is believed to involve multiple host, agent, and environmental factors. Which factors play the most significant roles and how those factors interact are poorly understood. A principal host factor is the composition of the gene that codes for the PrP protein. Genetic variabil- ity along the PrP gene sequence in animals suggests that evolutionary pres-

SURVEILLANCE FOR TSEs IN THE UNITED STATES 153 sure has led to the development of PrP sequences that are resistant to prion diseases. One of the best such examples is in sheep. Genetic resistance of sheep to the scrapie agent has been studied for decades. The polymorphisms at codons 136, 154, and 171 along sheep Prep have been recognized as major factors in determining resistance to scrapie. Three amino acids alanine (A), glutamine (QY, and arginine (R)- are encoded at those three sites. Sheep encoding ARR, especially those that are homozygous (ARR/ARR), are extremely resistant to scrapie. There has been only a single case of a sheep with an ARR/ARR genotype that devel- oped scrapie in nature. The sheep industry in the United Kingdom is taking active steps to inbreed ARR/ARR resistance into its flocks. Recently, how- ever, experimental i.c. inoculation of ARR/ARR sheep with the BSE agent led to the surprising observation that 3 of 19 sheep developed clinical symp- toms of TSE (Houston et al., 20031. This finding raises questions about the advisability of such inbreeding and suggests the need to investigate whether there is a difference in susceptibility to the scrapie and BSE agents in sheep. Much also needs to be learned about the genetic factors that determine susceptibility to human TSEs. We know that the PRNP gene in humans is polymorphic at certain locations; for instance, PrP may have either me- thionine or valine at codon 129. A recent report by Mead and colleagues (2003) suggests that human resistance to prion disease may be traced to early humans, whose practice of cannibalism placed them at risk of devel- oping fatal, kuru-like TSEs. The investigators hypothesized that this, in turn, resulted in selective survival of those resistant to TSEs. Individuals who are heterozygous at codon 129 of human PRNP appear to be more resistant to TSEs than those who are homozygous at that codon, according to this re- port, especially those who are homozygous for methionine. This hypothesis could explain the high frequency worldwide of heterozygosity at PRNP 129, the authors suggest, especially among the surviving female members of the Fore people who practiced ritual cannibalism (see Chapter 21. Distribution, Prevalence, and Host Range of TSEs The global distribution of TSEs is unknown because prion diseases could be occurring in areas where cases are unobserved or undiagnosed. Determining the extent of TSEs worIdwide will require not only better diagnostic tools, but also more extensive surveillance. Ecological research to improve sampling strategies, study animal movements and behavior, and develop nove! tracking methods would enhance the quality of TSE surveillance. It is likely that novel, undiscovered TSEs exist. They may be occurring in animals not previously known to have TSE, or known TSE agents may be presenting differently in new animal hosts. Surveillance systems must have

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