1 

## BSE Comprehensive Rule Supporting Scientific Documentation

August 2010



### Table of Contents

Executive Summary .................................................................................................1

I. Introduction .....................................................................................................2

II. Characteristics of the BSE Agent ...................................................................3

III. OIE Risk Classification Crite ria and Evaluation Process ...............................5

IV. Commodities for Trade .................................................................................14 Appendix ................................................................................................................35



### Executive Summary

The U.S. Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS) currently prohibits or re stricts the importati on of live ruminants and ruminant products

from certain countries 1 for bovine spongiform encephalopathy (BSE) based on regulations in title

9 of the Code of Federal Regulations sections 94.18(a)(1), (a)(2), and (a)(3). APHIS allows the importation of such commodities from countries not listed in those sections (assuming no other

restrictions due to other diseases are in place) because a risk from BSE risk is not evident. APHIS is proposing to amend these existing re gulations (specifically for bovines, bovine products, and bovine byproducts) by aligning them more closely with the 2009 recommendations from the World Organization for Animal Health (OIE).

APHIS intends to align its regulations with the OI E’s Terrestrial Animal Health Code (the Code) Chapter 11.6 (OIE 2009) by (1) adopting the categorie s and criteria for cla ssification of the BSE risk status of exporting countries, and (2) using those categories to determine appropriate import policies. For a country that has been evaluated and classified fo r BSE risk by the OIE, APHIS is proposing to base import policies on that classificati on. For a country that is not yet classified by the OIE as to BSE risk, APHIS would, upon reques t by the country, conduct a risk evaluation of the country, using criteria that are equivalent to those used by the OIE. This approach would improve the efficiency with which we could respond to export requests while continuing to protect U.S. cattle from exposure to BSE-in fected imports. Proposed changes would be

consistent with the most current scien tific understanding of BSE risk, based on the characteristics of the disease (including transmi ssion) and risk mitigations necessary to protect

animal health.

This supporting scientific documentation summarizes the significant compone nts of the relevant OIE BSE Chapter, including (1) the three levels of risk status classificat ion for countries, (2) the

1 Although APHIS’ BSE regulations refer to “regions” and the OIE BSE Code chapter refers to “countries, zones, or compartments,” for the sake of clarity, this document will refer to “countries ,” except when referring to BSE minimal risk regions.2 BSE country risk evaluation process, and (3) reco mmendations for restricted or unrestricted trade in certain commodities according to the BS E risk status of the exporting country.

Scientific literature and APHIS’ documentation in support of previous BSE regulations, as well as congruency between the evaluation processes of OIE and APHIS, demonstrate that aligning with the OIE Code is justified and effective

.



### I. Introduction

Developing science-based regulations that facilitate safe trade in cattle, beef, and beef products is a high priority for APHIS. APHIS’ BSE regul ations date back to 1989, and, in some cases, reflect a precautionary approach that was based on the more li mited scientific understanding of

the disease at the time. As new scientific findi ngs enhanced the knowledge of BSE, especially on the transmission of the disease and appropriate risk mitigations to minimize the potential transmission, APHIS periodically amended its regulations to reflect this knowledge. For example, in January 2005, APHIS amended the re gulations to establish a new BSE category for regions that pose a minimal risk of introducing BSE into the United States and to describe conditions for the importation of certain live ruminants and ruminant products and byproducts from such regions. In September 2007, APHIS agai n amended the BSE regulations to allow the importation of additional commodities from minimal risk regions. The risk assessments that accompanied these rules considered and evaluated risk mitigations imposed both in the country of origin and in the United States , recognizing that all of these risk factors must be considered together to appropriately evaluate the risk of establishment of BSE in the domestic cattle herd.

In addition to the amendments related to minimal risk regions, APHIS also amended the regulations in August 2005 to allow the importation of certain cuts of bone less beef from Japan. The risk assessment that accompanied that rule examined the evidence supporting the low risk associated with this commodity and was aligned with OIE’s findings related to deboned skeletal

muscle meat.

As knowledge of BSE increases, APHIS will conti nue to amend the BSE regulations whenever possible to ensure that trade policies are based on the most current science and are in step with international trade obligations.

In light of the greater scientific understanding of BSE, the OIE has revised its Code chapter on BSE, including adding a new classi fication structure (i.e ., negligible, controlled, or undetermined risk) to more accurately represen t the risk presented by countries for BSE. Based on scientific evidence, the OIE also stipulates different mitigations for those classifications that minimize the risk of BSE transmission. As a Member Country of the OIE, the United Stat es participates in the process of reviewing the BSE Code and is suppor tive of those changes that provide a sciencebased framework facilitating international trade.

This supporting scientific document summarizes th e significant components of the relevant BSE Code chapter and the scientific information th at supports the revised Code chapter. The key components include the three levels of risk status classification for countri es, the risk evaluation3 process, and the recommendations for restricted or unrestricted trade in certain commodities according to the BSE risk status of the exporting country.



### II. Characteristics of the Agent

BSE is a progressive neurological disorder of cattle that research suggests is caused by a pathogenic form of a normally occurring protein known as a prion (PrP) (Bolton et al. 1982; Prusiner 1994). BSE belongs to a family of diseases known as transmissible spongiform encephalopathies (TSEs). In addi tion to BSE, the TSEs include, among others, scrapie in sheep and goats, chronic wasting disease in deer and elk, transmissible mink encephalopathy, and Creutzfeldt-Jakob disease (CJD) in humans.

The pathogenic form of the prion protein (PrP Sc ) is both less soluble and more resistant to degradation than the normal form (T aylor 2000; Taylor et al. 1995). The PrP Sc is extremely resistant to heat and to normal sterilization pr ocesses, making it difficult to inactivate with standard methods used to process human f ood and animal feed. Although rendering and other processes can partially inactivate PrP Sc , the risk mitigation strategies (for meat and meat products) rely mainly on the elim ination of tissues and organs known to harbor BSE infectivity in infected animals, which are known as specified risk materials (SRM).

The removal of these specific tissues from the fo od chain eliminates more than 99 percent of the potential infectivity from inf ected carcasses, thereby prev enting consumer exposure through foods. Prohibiting their consumption provides a s ubstantial reduction in ri sk to consumers in

countries where BSE has been shown to exist an d in countries having a likely BSE risk. The

definition of SRM was first based on other TSEs (e.g., scrapie) and supplemented later with results obtained from the pathogenesis studies discussed in the appe ndix (Wells et al. 1994, 1996, 1998, and 1999).

Approximately 90 percent of the infectivity is as sociated with the brai n, spinal column, dorsal root ganglia, and trigeminal ganglia. The remain ing 10 percent is associated with the distal ileum. In BSE as in other TSEs, the total amount of infectivity in an an imal increases throughout the incubation period, reaching the highest load very close to the death of the animal. Infectivity is considered to increase exponent ially, reaching 3 logs less than a clinical case by 70 percent of the incubation period, and 4.5 logs less than a clin ical case at 50 percent of the incubation period (Comer and Huntly 2003). Consequently, what b ecomes an SRM is dependent on the age of the animal. The next paragraphs briefly describe in fectivity findings of SRM tissues at different stages of the incubation period.

 Brain – Studies in experimentally and naturally infected cattle demonstrate that the brain of BSE-infected cattle is exp ected to be infectious in the later stages of the incubation period (Wells et al. 1998 and 2005; European Commission Scientific Steering Committee [EC SSC] 2002a; Smith and Brad ley 2003; Hoffman et al. 2007).

 Spinal cord – Studies in experimentally and natura lly infected cattle de monstrate that the spinal cord of BSE-infected catt le is expected to be infectious in the later stages of the incubation period (Wells et al. 1998, 1999, a nd 2005; EC SSC 2002a; Smith and Bradley 2003; Hoffman et al. 2007).4  Tonsils –Results from experimentally infected cattle suggest that the palatine tonsil becomes infectious in the early stage of the incubation and that very low infectivity is maintained during the incubation period. In st udies where cattle were inoculated intra cerebrally (cattle bioassay) w ith palatine tonsils obtained from calves 10 months after they were orally infected with BSE, the cattle developed clinical evidence of BSE at 45 months post-inoculation. Therefore, the scientific community considers tonsils as SRM for cattle of all ages (Wells et al. 2005).

Intestine

– BSE infectivity has been found in the distal ileum, the jejunum, and the ileocecal junction of experimentally exposed cattle but not in ot her sections of the intestine. In addition, in naturally occu rring cases, sparse immunostaining has been observed in the myenteric plexus of the distal ileum, but not in thesubmucosal plexus of the distal ileum suggesting a very low level of infectivity in the enteric nervous system in the clinical phase of BSE (Terry et al. 2003) . Additionally, under field conditions, sparse immunostaining has been observed inconsistently in the distal ileum, the jejunum, and the

colon of a limited number of natural cases (Okada, 2010). A recent study of intestinal samples obtained from the German BSE pa thogenesis study (Hoffman et al, 2011) found high amounts of infectivity in the distal ileu m, moderate amount in ileocaecal junction and traces of infectivity in the jejunum. In contrast, the UK pathogenesis study (Stark, 2011) found that immunostaining of the intestinal nervous system was rare. In a different study of cattle challenged or ally with titred BSE br ainstem (Okada, 2011), PrP sc was confirmed in the distal ileum and the di stal jejunum by immu nohistochemistry and Western Blot. Infectivity in the ileum was reported after inoculation into transgenic mice.Since the enteric nervous system extends throughout the small intestine, the recent findings indicate that infectivity in animal s exposed to high doses of the agent could extend to the jejunum and the duodenum Give n that the cattle in the two pathogenesis studies described above were exposed to a ve ry high dose of infectivity that represents levels that exceed natural field exposure conditio ns, intestine other than the distal ileum is highly unlikely to contain epidemiologically sign ificant levels of infectivity to cause and perpetuate disease. Considering that the prevalence of BSE worldwide has been decreasing and given the fact that there is a linear correlation between prevalence and exposure, the likelihood that any cattle derive d jejunum could contain any infectivity is insignificant. Therefore the likelihood that a ny cattle could be exposed to any infectivity from the jejunum is extremely low; so low as to be considered ne gligible. Additionally, the international community has not changed it s definition of specified risk materials to include the jejunum based on the above findings . Since the distal ileum was found to be infectious early in the incubati on period, it is considered that the infectivity will remain in the tissue. Therefore, the scientific community considers distal ileum as SRM for cattle of all ages (Wells et al. 199 8 and 2005; Terry et al. 2003).

 Skull – Because of the skull’s association with brain and eyes, with resultant contamination through the slaughtering pro cess or because of residual brain tissue following removal of the brain, th e skull is considered to be SRM in the later stages of the incubation period.

 Vertebral column – Because of a combination of cl ose association with dorsal root ganglia and the superficial contamination of th e cut surface of the spine with spinal cord during the carcass splitting pro cess, the vertebral column is considered to be SRM in the later stages of th e incubation period.5 The agent does not evoke a traditional im mune response or inflammatory reaction (Khalili-Shirazi et al. 2005); t hus, reliable ante-mortem diagnos tic tests based on host reaction

are not available. Definitive diagnosis requires post -mortem microscopic examination of brain

tissue or detection of PrP Sc in tissue samples.

In the past few years, cases of BSE have been identified throughout the world in which various biological features differ, such as molecular characteristics of the abnormal prion protein, absence of the spongiform changes in the brain, or different location of the spongiform lesions (Béringue et al. 2006; Buschmann et al. 2006a; Ca salone et al. 2004; De Bosschere et al. 2004; Buschmann et al. 2006; Lloyd et al. 2004; Yamakawa et al. 2003) . These cases have generally been referred to as “atypical BSE,” although the literature describes seve ral different forms of unusual cases. To date, however, the understanding of the risk of transmission of BSE (and/or the SRM definition) remains unaltered, even when incorporating these recently identified unusual or so-called “atypical” BS E cases (OIE 2003). The appendix

, as well as APHIS’ peerreviewed risk assessment that accompanied the most recent BSE rulemaking related to minimal risk regions (APHIS 2007) describes other characteristics of the agent such as epidemiology, transmission studies, and pathogenesis.



### III. OIE Risk Classification Criteria and Evaluation Process

The OIE is the international organization recognized by the Wo rld Trade Organization as the standard-setting body charged with developing science-based r ecommendations for trade in animals and animal products. The OIE publishes its standards in the Terrestrial Animal Health Code. Unless there is scientific justification fo r not doing so, Member Co untries are required to base their import health measures on international standards.

Within the last few years, the OIE has revised its Code chapter on BSE (OIE 2009) by adding a new classification structure to more accurately represent the risk presented by countries. The 2009 OIE guidelines provide for three possible BSE ri sk classifications for countries: negligible risk, controlled risk, and undetermined risk (Articles 11.6.3, 11.6.4, and 11.6.5). This framework not only recognizes different levels of risk amon g countries but also establishes risk mitigation measures for trading in live an imals and products, even from count ries considered undetermined

risk.

As described Articles 11.6.2 and 11.6.23 to 11.6.28 of the Code, countries requesting BSE

classification from the OIE must submit a dossi er containing information to support a risk assessment. OIE uses the information to complete a release assessment (which assesses the likelihood of introducing BSE into the exporting country through live cattle, products derived from cattle, or meat-and-bone meal) and an exposure assessment (which considers the likelihood that cattle within the exporting country coul d have been exposed to the BSE agent through factors such as feeding practices, the use of bovine carcasses and slaughterhouse waste, rendering methods, and the enforcement of an ef fective ruminant-to-ruminant feed ban). A requesting country must also submit informa tion on its awareness program and notification requirements for BSE, its diagnostic capabilit ies, and its BSE surveillance program. The information in this dossier is evaluated by lead ing BSE experts from OIE member countries to6 determine the country’s BSE risk status cla ssification, which in turn will determine import requirements for live cattle and cattle products th at are not designated fo r unrestricted trade.

To retain OIE’s classification of disease stat us, a country must continue to observe OIE’s guidelines and report any signifi cant events that might change that status (OIE 2006). OIE reserves the right to re voke the disease status of any country that fails to comply with this process. Member Countries must notify OIE in writing by November of each year that the epidemiological situation with respect to each of the diseases for which OIE has classified the country has remained unchanged.

The OIE recommends that after the occurrence of a BSE case in a negligible-risk country, the country should be classified as controlled BSE ri sk unless the criteria of Article 11.6.3 continue to be met. For reinstatement of negligible ri sk status, the country should submit a dossier of explanatory data to the OIE as quickly as possible and at the la test by the November deadline for

the submission of annual risk assessment data.

As described in the OIE Code Articles 11.6.3 to 11.6.5, certain criteria must be met for each of

the risk classifications.

Negligible BSE risk

Countries defined as negligible risk must have de monstrated that appropriate measures have been taken for a specific time and have managed th e risks. Live cattle and cattle commodities originating from negligible-risk countries are cons idered to pose a negligible risk of transmitting the BSE agent to the importing country.

The following criteria must be met to be classified as negligible risk:

1. A risk assessment has been conducted in order to identify the historical and existing risk factors, and the country has dem onstrated that appropriate speci fic measures have been taken for the relevant period of time defined below to manage each identified risk;

2. The country has demonstrated that an adequa te surveillance program is in place; and

3. EITHER:

a. There has been no case of BSE identified with in the country, or, if there has been a case, every case of BSE has been demonstrat ed to have been imported and has been completely destroyed, and

i. Appropriate awareness and notificati on programs and laboratory diagnostic procedures have been in place for at least 7 years; 2 and

ii. It has been demonstrated through an appr opriate level of control and audit that for at least 8 years 3 neither meat-and-bone meal nor greaves derived from ruminants has been fed to ruminants;

2 The 7-year requirement reflects the 95 th percentile of the normal distribution of the age of clinical BSE cases detected at the peak of the United Kingdom and Swiss epidemic.

3 Because of the demonstrated risk of MBM, one year was added as a precautionary measure.7 OR

b. If there has been an indigenous case, every indigenous case was born more than 11 years ago; and

i. Appropriate awareness and notificati on programs and laboratory diagnostic procedures have been in place for at least 7 years; and

ii. It has been demonstrated through an appr opriate level of control and audit that for at least 8 years neither meat-and- bone meal nor greaves derived from ruminants has been fed to ruminants; and

iii. All BSE cases, as well as:

 All cattle which, during their first year of life, were reared with the BSE cases during their first year of life, and which investigation showed could have consumed the same potentially contaminated feed during that period, or

 If the results of the investigation are inconclusive, all cattle born in the same herd as, and within 12 months of the birth of, the BSE cases, if alive in the country, are permanently identified, and their movements controlled, and, when slaughtered or at death, are completely destroyed.

Controlled BSE risk

Countries defined as controlled risk must have demonstrated that appr opriate measures have been taken to manage any risks identified, but not for the time described in the OIE Code for negligible risk countries. Certain conditions ar e necessary for trade in live cattle or cattle products from a country desi gnated as controlled risk.

The following criteria must be met for a count ry to be designated as controlled risk:

1. A risk assessment has been conducted in order to identify the historical and existing risk factors, and the country has demonstrated th at appropriate measures are being taken to manage all identified risks, but these measures have not been taken for the relevant period of time;

2. The country has demonstrated that adequa te surveillance has b een carried out; and

3. EITHER:

a. There has been no case of BSE or, if ther e has been a case, every case of BSE has been demonstrated to have been impor ted and has been completely destroyed, appropriate awareness and notificati on programs and laboratory diagnostic procedures are in place, and it can be dem onstrated through an appropriate level of

control and audit that neither meat-a nd-bone meal nor greaves derived from ruminants has been fed to ruminants, but at least one of the following two conditions applies:

i. The awareness and notification pr ograms and laboratory diagnostic procedures have not been in place for at least 7 years;8 ii. It cannot be demonstrated that cont rols over the feedi ng of meat-and-bone meal or greaves derived from ruminants to ruminants have been in place for 8 years;

OR

b. There has been an indigenous case of BSE, awareness and notification programs and laboratory diagnostic procedures are in place, and it can be demonstrated through an appropriate level of control and audit th at neither meat-and-bone meal nor greaves

derived from ruminants has been fed to ru minants, but at least one of the following two conditions applies:

i. Awareness and notification programs a nd laboratory diagnostic procedures have not been in place for at least 7 years;

ii. It cannot be demonstrated that cont rols over the feedi ng of meat-and-bone meal and greaves derived from ruminants to ruminants have been in place for 8 years;

AND

iii. All BSE cases, as well as:

 All cattle which, during their first year of life, were reared with the BSE cases during their first year of life, and which investigation showed could have consumed the same potentially contaminated feed during that period, or

 If the results of the investigation are inconclusive, all cattle born in the same herd as, and within 12 months of the birth of, the BSE cases, if alive in the country, are permanently identified, and their movements controlled, and, when slaughtered or at death, are completely destroyed.

Undetermined BSE risk

The cattle population of a country poses an undeterm ined BSE risk if it cannot be demonstrated that it meets the requirements of another ca tegory. Either the country has undergone the evaluation process and has not met the conditions fo r either negligible or controlled risk status, or it has not submitted sufficient information (or any information) to be evaluated.

Risk assessment and other requirements for classification

The OIE recommends that a risk assessment be conducted to support classi fication for controlled and negligible risk (Article 11.6.2; OIE 2009). The risk assessment is the primary tool on which

the classification of BSE status is based. The OIE recommends that the risk assessment identify

all risk factors relevant to BS E occurrence in the country. A rel ease assessment and, if justified, an exposure assessment may be part of that risk assessment.

The release assessment should address the likelih ood that the BSE agent al ready exists or has been introduced into the exporti ng country through the importation of potentially infective cattle or contaminated cattle products.9 The exposure assessment should as sess the likelihood of exposure of the BSE agent to cattle in the exporting country. The exposure assessment s hould include information on the following:

 the likelihood of recycling and amplificat ion of the BSE agent through consumption by

cattle of meat-and-bone meal or greaves of ruminant origin or other feed or feed ingredients contaminat ed with these;

 the origin and use of cattle carcasse s (including fallen st ock), byproducts, and slaughterhouse waste;

 the parameters of the rendering processes, ca ttle feeding practices , and the methods of cattle/animal feed producti on in the exporting country;

 the potential for the exposure of cattle to the BSE agent through consumption of meatand-bone meal or greaves of ruminant origin; and

 the implementation and enforcement of feed bans, including measures to prevent crosscontamination of animal feed.

Countries also need to submit information about the presence of an awareness program and notification requirement for BSE, the presence of an adequate BSE surveillance system, adequate diagnostic capability, and the histor y of BSE in the exporting country.

Release assessment of the exporting country

The exporting country’s release as sessment addresses the potential for the release of the BSE agent into the country through the importation of:

 Meat-and-bone meal or greaves;

 Potentially infected live cattle; and/or

 Products of animal origin potentially infected with BSE.

Meat-and-bone meal or greaves

It is widely accepted in the scientific community that BSE is caused by the consumption of meatand-bone meal or greaves of ruminant origin that contain the BSE agent. Therefore, to assess the risk of the release, the expor ting country must have knowledge of the origin of meat-and-bone meal, greaves, or feedstuffs imported into the country. Meat-and-bone meal and greaves originating from high-risk countries for BSE presen t a higher release risk than those originating from low-risk countries. Exporting countries mu st provide information about whether meat-andbone meal, greaves, or feedstuffs containing any of these commodities have been imported into that country within the past 8 years, and, if so, from what c ountry and in what quantities.

Potentially infected live cattle

Assessing the risk of the rele ase of the BSE agent in the exporting coun try through the importation of potentially infect ed live cattle into the country relies on several underlying assumptions (OIE 2009). The most significant of th ese is that the risk of BSE transmission is influenced by the date at which imports occurred (relative to the BSE m itigations in the country of origin) and is proportiona l to the volume of potentially contaminated imports.10 Based on these assumptions, the risk of the rele ase of the BSE agent in the exporting country depends on the following:

 The BSE status of the country of origin of the potentially infected live cattle; however, the BSE status could change as more data become available, including the detection of clinical disease or following active surveillance;

 The species of imported animals, in this case, cattle;

 The feeding and management of the cattle in the country of origin;

 Whether the cattle are dairy or meat breeds, if there are differences in exposure due to feeding practices in th e country of origin;

 Whether the bovine animals (including bison) were imported for breeding or slaughter;

and

 Age at slaughter.

The exporting country must provide information with in the context of these risk factors about the importation of live cattle duri ng the previous 7 years.

Products of animal origin poten tially infected with BSE

To assess the risk of the releas e of the BSE agent from an expor ting country, OIE must consider the importation of animal products that could be infected with BSE. As with importation of potentially infected live cattle, the underlying assumptions regarding the date and volume of imports affect this assessment.

Based on these assumptions, the risk of the release of the BSE ag ent in the exporting country is dependent on:

 The species of origin of the animal products and whether these pr oducts contain tissues known to contain BSE infectivity.

 Country of origin and its BSE status. However, the status could change as more data become available, including the detection of clinical disease, or following active

surveillance.

 Feeding and management of the an imals in the country of origin.

 Use of the commodity. Apart from representing the risk of developing clinical disease, the slaughter, rendering, and recycling in meat-and-bone meal of imported animals represent a potential route of exposure for indigenous lives tock even if meat-and-bone meal and greaves, or feedstuffs cont aining them, have not been imported.

 Whether the cattle in the country are dairy or meat breeds, if there are differences in exposure due to feeding practices in the country of origin.

 Age at slaughter.

The exporting country provides information, within the context of the risk factors listed above, about the importation of produc ts of animal origin duri ng the previous 7 years.

Exposure assessment of the exporting country

The exposure assessment assesses the likelihood of exposur e of the BSE agent to cattle in the exporting country. The exposure assessment should include information on the potential for the11 exposure of cattle to the BSE agent through cons umption of meat-and-bone meal or greaves of bovine origin; the implementation and enforcement of feed bans, including measures to prevent

cross-contamination of animal f eed; the recycling and amplification of the BSE agent; the origin and use of cattle carcasses (including fallen stock), byproducts, and slaughterhouse waste; the parameters of the rendering processes; and the methods of cattle/a nimal feed production.

The potential for the exposure of cattle to the BSE agent thro ugh consumption of meat-and-bone meal or greaves of bovine origin

According to scientific litera ture, cattle consumption of m eat-and-bone meal or greaves of ruminant origin plays the main role in BSE tran smission. If cattle have not been fed products of bovine origin (other than milk or blood) potentially containing meat-and-bone meal or greaves of bovine origin for the past 8 years, exposure to BSE is extremely unlik ely. (Countries applying for negligible risk status are required to demonstrate that th e ruminant feed ban has been effective for at least 8 years and all BSE cases were imported or, if indigenous, were born more than 11 years ago.)

Exporting countries provide information on whether meat-and-bone meal or greaves of bovine origin have been fed to cattle in the country within the previous 8 years, including a discussion of the implementation and enforcement of feed bans, including measures to prevent crosscontamination of animal feed.

The origin of cattle carcasse s, by-products, and slaughterhouse waste; the parameters of the rendering processes; and the met hods of cattle feed production

Evidence indicates that the transmission of BSE re quires that cattle ingest feed that has been contaminated with tissue from an infected animal carrying the BSE agent. Several steps must take place for this to happen: an infected anim al, carrying significant amounts of the infectious agent, must die or be slaughtered; tissues from th at animal that contain th e infectious agent must be sent to a rendering facility; the infectivity pres ent in these tissues must survive inactivation in the rendering process; the resulting protein must be incorporated into feed; and this feed must be fed to at least one bovine at an adequate level.

The rendering industry is crucial in reducing th e risk of transmitting BSE infectivity, not only

because of its role in inactivati on of the BSE agent but also because it serves as a critical control point for the redirection of ruminant protein aw ay from cattle feeds. If a fraction of the BSE infectivity were to escape destru ction at the rendering facility, it would need to bypass controls imposed to prevent cross-contamin ation and ensure proper labeling of rendered materials (at the renderer) and feeds produced using prohibited meat-and-bone meal (at the feed mill).

The overall risk of BSE in th e cattle population of a country is proportional to the level of known or potential exposure to BS E infectivity and the potential for recycling and amplification of the infectivity through livestock feeding practices. If potentially infected animals or contaminated materials are rendered, there is a risk that the resulting meat-and-bone meal could retain infectivity.12 For the risk assessment to conclude that the cat tle population of a country is of negligible or controlled BSE risk, it must have demonstrated that appropriate measures have been taken for a set period of time to manage any risks. Countries must provide information regarding the ways in which cattle carcasses, cattle bypr oducts, and slaughterhouse waste have been processed over the previous 8 years in the country.

Additional factors to be considered in the determination of BSE risk status

Epidemiological situation of BSE in the country

OIE assesses the epidemiological situation by evaluating the BSE hist ory of the country, including surveillance data. Surv eillance programs generate a picture of the epidemiological situation of BSE in the country (Article 11.6.22). The more targeted the surveillance activities, the more meaningful the information. Surveillan ce targeted at high-risk populations for BSE provides better information than generic animal disease surveillance for the entire cattle population (USDA 2006).

BSE surveillance

Depending on the characteristics of the countr y, the goal of BSE surveillance could be to:

 Detect cases at a predeter mined design prevalence;

 Monitor the evolution of the disease (i.e., intr oduction and/or spread);

 Monitor the effectiveness of a feed ban a nd/or other risk mitigation measures; and

 Provide supporting evidence for claimed BSE status, for maintaining status, or for advancing to a higher status.

Targeted sampling for BSE surveillance focuses on two factors that have been shown to be relevant to determining the risk for BSE: clinical presentation and age. For the purpose of disease detection, it is most efficient to conduct targeted surveillance by collectin g as many samples as possible from the surveillance stream that ha s the greatest likelihood of having the disease

 that is, cattle displaying clinical signs consistent with BSE. Since it is likely that a country cannot meet its needed number of samples from this surveillance stream alone, additional samples

should be taken from the surveillance streams wi th the next highest li kelihood for finding BSE: animals that are nonambulatory or condemned ante -mortem at slaughter (casualty or emergency slaughter), followed by cattle which are found dead (f allen stock). Of lowest priority are healthy slaughter cattle.

The OIE Code on BSE surveillance (Article 11.6.22) reflects international scientific consensus and provides guidelines for surveillance programs based on a weighted point system. The highest point values are assigned to those samples from animals with classic clinical signs of the disease; the lowest point values correspond to clinically normal animals tested at routine slaughter. The goal of this weighted approach is to ensure that countries sa mple those cattle populations where the disease is most likely to be found (USDA 2006).

OIE has identified the following four subpopulati ons of cattle for su rveillance purposes:13 1. Clinical suspects: Cattle over 30 months ol d displaying behavioral or clinical signs

consistent with BSE.

2. Casualty slaughter: Cattle over 30 months, nona mbulatory, recumbent, unable to rise or to walk without assistance, sent for emerge ncy slaughter or condemned at ante-mortem inspection.

3. Fallen stock: Cattle over 30 m onths and found dead on-farm or during transport to or at

an abattoir.

4. Healthy slaughter: Cattle over 36 months exhibi ting no clinical signs consistent with BSE

or other diseases

Type A surveillance. The OIE BSE surveillance guidelines recommend a target number of surveillance points for Type A surveillance based on the size of a country’s adult cattle population. For instance, a c ountry with an adult cat tle population of 800,000 to 1,000,000 should collect samples whose total value equals 240,000 points. These points are accrued over 7 consecutive years and are weighted according to the surveillance stream and age of the animal sampled. This type of surveillance is recomm ended for countries that would like to meet

controlled risk status.

Type B surveillance. Type B surveillance may be carried out by countries of negligible BSE

risk status to confirm the c onclusions of the risk assessment; a country can demonstrate the effectiveness of risk mitigation measures by targeting surveillance to find failures in those mitigations.

Controlled-risk countries may al so conduct Type B surveillance af ter meeting the points target using Type A surveillance to maintain confiden ce in the conclusions reached through Type A surveillance. As with Type A surveillance, the recommended number of points for Type B

surveillance is based on the size of a country’s cattle population. For instance, a country with 800,000 to 1,000,000 adult cattle should collect sa mples whose total point value equals 120,000.

Presence of a BSE awareness program

Countries must provide to the OIE information about the existence of any awareness programs, including the target audience, th e curriculum, how long the program has been in place, and any contingency and/or preparedne ss plans that address BSE.

Compulsory notificati on and investigation

The socioeconomic implications associated with BSE require that there be incentives and/or obligations to report and investigate suspect cases. Therefore, c ountries must provide information about guidance given to veteri narians, producers, workers at auctions, slaughterhouses, etc. with regard to the criteria that w ould initiate the investig ation of an animal as a BSE suspect; whether these criteria have ch anged over time; the date and content of the legal act making notification of BSE suspects compulsory; and any measures in place to stimulate notification, such as compensation paym ents or penalties for not reporting a suspect.14 Sample testing

For a country’s BSE surveillance system to be rec ognized by the OIE, samples must be tested in

accordance with the OIE’s Manual of Diagnostic Tests and Vacci nes for Terrestrial Animals (OIE 2009b). Exporting countries must provide information about whether the diagnostic procedures and methods used in the country are consistent with those described in the manual and whether these diagnostic procedures and methods have been applied through the entire surveillance period.

BSE history of the country

Countries must provide general information about whether BSE has occurred in the country, and, if so, how it has been addressed. In addition, to retain OIE’s clas sification of disease status, a country must continue to observe OIE’s guidelines and report any significant events that might change that status. The OIE rese rves the right to revoke the give n disease status of any country that fails to comply with this process. In November of each year, Member Countries are obligated to notify the OIE in writing that the ep idemiological situation with respect to each of

the diseases for which the OIE has classified the country has remained unchanged to retain

classification.



### IV. Commodities for Trade

OIE bases import requirements for live cattle an d cattle products on two criteria: the risk

classification status of the e xporting country and the risk of BSE transmission posed by the various products. This section discusses the ri sk associated with specific commodities with regard to BSE transmission, incl uding experimental data, epid emiological data, information about risk mitigation through processing, data from risk assessment studies, and a discussion of potential BSE infectivity for each commodity, where available. Following each discussion, we present OIE’s conclusions about th e risk associated with trade of the specific commodity as well as any conditions necessary for trade based on the risk classificat ion of the country.

Commodities for unrestricted trade

OIE has recognized that certain products can be traded regardless of the BSE status of the exporting country, zone, or compartment (OIE 2009). These products, defined as unrestricted commodities, should be allowed to be imported re gardless of the BSE risk classification of the exporting country:

 Semen and in vivo derived embryos;

 Gelatin and collagen from hides and skins;

 Tallow with a maximum level of insolubl e impurities of 0.15 percent in weight and derivatives made from this tallow;

 Dicalcium phosphate with no trace of protein or fat;

 Deboned skeletal muscle meat from cattle (under certain conditions);

 Blood and blood by-products (under certain conditions);

 Milk and milk products; and15  Hides and skins. APHIS regulations do not currently prohibit or restrict the importation of milk and milk products, semen, or hides and skins. Because no amendments to the existing regulations are required for these products, this risk document will not discuss the relevant scientific literature; however, these commodities are included on the list fo r completeness and to align with the list in the 2009 OIE Code. Although APHIS regulations do not prohibit or restrict the importation of in vivo derived embryos, certain cer tification requirements are in place through separate import protocols. Therefore, the scien tific literature relevant to this commodity is discussed here.

Commodities for restricted trade

Other commodities that have been shown to pos e a potential risk for BSE transmission may or may not be allowed to be imported or may require different mitigations, based on the risk posed by the individual commodity according to the BSE risk classification of the exporting country.

These commodities should be allowed for restricted trade:

 Live cattle;

 Meat other than deboned skeletal muscle meat, meat products, and meat byproducts under certain conditions;

 Gelatin and collagen prepared from bones and intended for food, animal feed, fertilizers, veterinary biologics, or as unpr ocessed material for the prep aration of cosmetics, drugs (including pharmaceuticals and natural hea lth products) or medical devices;

 Tallow and tallow derivatives (other than ta llow with a maximum level of insoluble impurities of 0.15 percent in weight) and dicalcium phosphate with fat intended for food, animal food, fertilizers, cosmetics, pharm aceuticals including biologicals, or medical devices; and

 Meat-and-bone meal.

Specified risk materials (SRM)

Conditions for trade in several of the restrict ed commodities include removal of SRM. These materials pose the greatest risk of containing th e BSE agent according to the OIE: (1) tonsils and distal ileum from animals of all ages, (2) brains , eyes, spinal cord, skull, and vertebral column from animals 30 months of age or older from controlled-risk countries , and (3) brains, eyes, spinal cord, skull, and vertebra l column from animals over 12 mont hs of age from undeterminedrisk countries. Although the skul l and vertebrae themselves do not carry infectivity, they are

considered risk materials because of th eir association with infective tissues.

Similarly, in the United States, the Food Safety a nd Inspection Service (FSIS) has regulations to protect human health that also address animal health issues . In 2005, FSIS amended the Federal meat inspection regulations to prohibit the us e of SRM for human food (FSIS 2005). This rule established as SRM the skull, brain, spinal cord, trigeminal ganglia, eyes, vertebral column (excluding the vertebrae of the tail, the transverse processes of the thoracic and lumbar vertebrae, and the wings of the sacrum), and dorsal root ganglia of cattle 30 months of age or older, as well

as the tonsils and distal ileum of the small intestine of cattle of all ages.16 Unlike other parts of the vertebra l column, the vertebrae of the ta il, the transverse processes of the thoracic and lumbar vertebrae, and the wings of the sacrum do not contain spinal cord or dorsal root ganglia. Therefore, FSIS excluded these parts of th e vertebral column from the materials designated as SRM.

APHIS is proposing to adopt the FSIS definition of SRM and apply it to cattle 30 months of age

or older for controlled-risk c ountries and to cattle 12 months or older for undetermined-risk countries. This age-based approach is consistent with OIE guidelines. Research demonstrates that the incubation period for BSE is linked to th e infectious dose received, i.e., the larger an infectious dose received, the shorter the in cubation period (EC SSC 2002a). Few cases of BSE

have been found in animals under 30 months of age, and they have occurred primarily in countries with significant levels of circulating inf ectivity. Specifically, BSE was found in animals under 30 months of age in the UK in the late 1980s to early 1990s, when the incidence of BSE was extremely high; the youngest case, detected in 19 89, was 21 months. The detection of BSE in young animals during the peak of th e BSE epidemic in the UK supports a cautious approach in defining SRMs for regions of undeterm ined risk for BSE. Theoretically, in such regions, high levels of circulating infectivity could exist if the region is not conducting BSE surveillance or implementing BSE risk manage ment measures. Because BSE infectivity is

detectable in central ne rvous system (CNS) tissue at three-quarters of the incubation period, the 12-month provision would ensure the removal of tissues potentially containing infectivity from even the single youngest animal observed sinc e the start of BSE su rveillance in the UK.

In vivo derived cattle embryos

Supporting documentation

. No detectable infectivity has been found in susceptible mice fed placenta from confirmed cases of BSE (Mi ddleton and Barlow 1993; Barlow and Middleton 1990; Bradley 1990), nor in placenta, placental flui ds, or ovary or uter ine caruncle following mouse inoculation (Fraser and Fo ster 1994; Ministry of Agriculture, Fisheries, and Food 1997; EC SSC 2002b). Male reproductive tissues (testis, epididymis, prostate, semen, seminal vesicle) inoculated into mice also showed no infectivity (Fraser and Foster 1994). In addition, infectivity was not detected in the fetal membranes and placent a of cattle with clinical BSE after cattle were dosed oronasally with a pooled tissue homogenate from BSE cattle. Animals were killed at 24 and 84 months post infection with no evidence of disease (Bradley 1996; EC SSC 2000b).

A different study failed to detect infectivity in semen of BSE-infected bulls (Wrathall et al. 2002). In this study, semen from 13 bulls (8 wi th clinical BSE) wa s used for artificial insemination of 167 clinically a ffected cows in the terminal stages of BSE. The resulting embryos were treated according to the recomm endation of the International Embryo Transfer Society (IETS). The embryos were always transfe rred singly, but repeat transfers were done if

returns to estrus occurr ed within the particular transfer session. Researchers transferred 587 viable embryos into 347 recipient heifers im ported from New Zealand. A total of 266 live offspring were born, of which 54.1 percent had a BSE-positive sire as well as a BSE-positive dam. The recipient heifers were monitored for clinical signs of BSE for sev7en years after transfer, and the offspring were monitored for 7 years after birth. Twen ty-seven heifers and 20 offspring died during monitoring but none showed si gns of BSE. The brains of these animals, in

addition to the brains of animals killed after 7 years, were examined for BSE by histopathology, by PrP immunohistochemistry, and by electron microscopy for scra pie-associated fibrils. All17 results were negative. In addition to the embr yos transferred into re cipient heifers, 1,020 nonviable embryos were inoculated intracerebra lly into susceptible mice (20 embryos per mouse) that were monitored for up to 700 days post inoculation; their brains were then examined for spongiform lesions. All results were negative. Additionally, uterine flush fluid samples from 41 cows were tested for BSE infectivity by intr acerebral and intraperito neal inoculation of 946

mice. One of these mice had some vacuolar pathology, but its relevance proved difficult to determine because the putative incubation period wa s inconsistent with the survival of remaining mice in the group. All other mice with injections of flush fluids from the same cow were negative when finally killed and examined. Resu lts of the study indicate that embryos are unlikely to carry BSE and do not transmit the dise ase to recipients and their embryo transfer offspring, even when they are collected from don or cows at the end-stage when the risk of maternal transmission (if it were to ex ist) could potentia lly be the highest.

In a cohort study (Wilesmith et al. 1997), 316 offspring of BSE confirmed cows (cases) and 316 offspring from cows over 6 years old and wit hout BSE from the same farm and age cohort (controls) have been observed under controlled c onditions over a 7-year period. The purpose of the study was to determine whether maternal tran smission occurs, and, if so, at what level of

incidence. There was a statistical ly significant risk difference be tween the two cohorts examined (i.e., calves born to dams with BSE and calves born to healthy dams more than 6 years old). This difference was 9.7 percent with a re lative risk of 3.2 for offspring of cows that developed clinical BSE. This enhanced risk for the offspring of BSE dams appeared to decline the later the offspring were born after the 1988 feed ban was in place but increased as parturition was closer

to the onset of clinical diseas e in the dam. The results cannot distinguish between a genetic component and true maternal transmission, for which there is no other evidence. Instead, a combination of a genetic cause (i.e., increased su sceptibility to feed exposure that could have occurred in any cattle in the st udy) and genuine transmission best fits the computer model of the epidemic (Donnelly et al. 1997). Later studies by Donnelly et al. (2002) si gnificantly reduced the estimated risk to offspring, although the authors recognized that the intr oduction of culling of offspring of confirmed cases made estimation of the risk impossible other than by backcalculation methods. The route for the hypothetical maternal transmission of BSE has not been established. Based on the modelling study, given that less than one percent of the offspring of affected cattle in the UK epidemic may succumb to this means of exposure, it is likely to be

difficult to determine the route.

More recent work on cases born after the 1996 feed ban fails to demonstrate evidence of maternal transmission (Hill 2005). Thus, although ma ternal transmission may be possible, more recent epidemiologic evidence suggests that maternal transmission of BSE is unlikely to occur at any appreciable leve l, if at all.

OIE guidelines

. Experimental and epidemiological evid ence do not support transmission of the BSE agent through male and female reproductive tissues. Therefore, OIE concludes that the trade of in vivo derived cattle embryos coll ected and handled in accordance with the

recommendations of the IETS should be unrestr icted, regardless of the BSE status of the exporting region.

Gelatin18 Supporting documentation. Gelatin is a highly purified protein ma nufactured from hides, skin, and/or bones of animals using various refining processes in which each step is able to significantly inactivate any BSE infectivity. The st eps of the refining process, as well as the resulting infectivity reduction, ar e described below (Article 11.6.15).

1. Degreasing : Before bone can be used to manufacture gelatin, fat must be removed. This is done by crushing the bones and washing and degreasing the chips with hot water to remove fat residues. Studies evaluating the efficiency of the degreasing process in decreasing the amount of nervous tissues pr esent in bones have shown that 98–99 percent of the proteins of nervous origin are eliminated (Mantze et al. 1996).

2. Acid treatment : The treatment consists of immersing the degreased bone chips into hydrochloric acid (approximately 4 percent, pH <1.5) for at least 2 days. This acid treatment changes the structure of the collage n protein and is belie ved to decrease the infectivity titer that might be present (Grobben et al. 2004).

3. Alkaline treatment : The materials are soaked in a sa turated lime solution (pH 12.5) for 20 to 50 days. The alkaline treatment changes th e internal structure of the BSE protein, if present. The combination of time, temperat ure, and concentration of the alkaline

treatment reduces the titres of BSE infectivit y if present in the ra w materials (Grobben et al. 2004).

4. Further acid treatments : In the event gelatin is produced from ossein by an acid process,

the ossein is immersed for 12 to 24 hours in acid (pH 2 – 3.5).

5. Gelatin extraction : Once all the procedures are performe d, gelatin is extracted by a series of hot water steps. These include purification by filtration and sterili zation, both of which further remove suspended materials and thus further reduce the level of any remaining BSE infectivity, if present, which is un likely at this stage in the production.

Research studies mimicking the manufacturing pr ocess described above were unable to show detectable levels of infectivity in the mouse bioassay. The results are consistent with TSE infectivity reduction capacity, ex ceeding a factor of 30,000 (4.5 logs , although results from most

recent research indicate clearance factors exce eding 4.8 logs) (EC SSC 2003a). These studies have demonstrated that the common proce ss of manufacturing bovine gelatin provides significant assurance of low risk for BSE.

A quantitative risk assessment by the Europ ean Food Safety Authority (EFSA 2006) of the residual risk in bone-derived gelatin (obtained from bones fit for human consumption) calculated different scenarios resulting in diffe rent risk levels. The study did not take into consideration the sourcing of bones. Results indicate that the relevant exposures ar e very small compared to the historical exposure of the human population in the UK ( 1980–2001) due to meat and meat products in the diet. The removal of skull and ve rtebral column from the source materials results only in a very small risk reduction. However, the input parameters to the supporting risk assessment model sourced animals only from th e healthy slaughter s ubpopulation and did not

address the scenario where material was sourced from cattle not subject to ante- and post-mortem inspection. This evidence points to the conclusion that bone-deriv ed gelatin that is produced using common manufacturing processe s could be considered safe re gardless of the country from which the bones originate.19 OIE guidelines. Gelatin and collagen prepared from hides and skins are not risk factors in transmitting BSE. However, OIE considers that ge latin and collagen derived from bones can be traded based on manufacturing prac tices and the following conditions:

1. Negligible-risk countries: Gelatin and collage n derived from bones can be traded freely provided that cattle pass ante-mor tem and post-mortem inspection.

2. Controlled and undetermined-ris k countries: Gelatin and coll agen derived from bones can be traded provided that (1) cattle passed an te-mortem and post-mortem inspection; (2) the

vertebral column from cattle ove r 30 months of age at the time of slaughter and skulls are excluded from processing; and (3) the bones ar e subjected to the pr ocess listed in OIE Code Article 11.6.15 or equivalent.

Tallow

Supporting documentation. Several studies have ev aluated TSE infectivity in tallow that was spiked with infected brain materi al and then subjected to render ing. In two rendering studies, one with BSE (Taylor et al. 1995) and the other wi th scrapie (Taylor et al. 1997), no detectable infectivity to either agent was demonstrated fr om any of the tallow samples when assayed in mice. The BSE rendering study did not demonstrat e any infectivity in crude unfiltered tallow, although the same rendering procedure produced meat-and-bone meal with almost the same infectivity titer levels as th e untreated raw material. These studies suggest that during the manufacturing process of tallow, both BSE and scra pie agents do not preferentially separate with tallow during rendering but tend to remain with the meat-and-bone meal fraction.

A recent review of inactivation of TSE agen ts during rendering (Taylor and Woodgate 2003) suggests that tallow is generally not considered to pose a risk of BSE infection for two main reasons: (1) BSE-spiked rendering st udies confirmed the lack of detectable infectivity of tallow through mice bioassay, and (2) epidemiological studies were not able to li nk the distribution and

use of tallow in cattle feed to the incidence of BSE in the UK.

Some countries (such as Denmark and Japan) have implicated tallow in milk replacers as a potential source of BSE infection. A 2003 epidem iological report on BSE in Japan hypothesized that tallow in calf milk replacer was one po ssible source and route of infection. However, statistical analysis of the data did not support any correlation between th e use of milk replacer and BSE incidence (Ministry of Agriculture, Forestry, and Fi sheries 2003). A more recent retrospective study of BSE cases in Japan implicated milk replace rs, produced by a single factory containing both domestically produced tallow and tallow imported from the Netherlands, in a

cluster of 13 calves later confirmed to have BSE (Tsutsui et al. 2008). Calf concentrates produced by the same factory, and which could have been contaminated with meat-and-bone meal, were also fed to the cluste r, thus leading the au thors to conclude that the association with calf concentrates could have been masked in the analysis.

A quantitative risk assessment of BSE transmission through tallow- based milk replacer (Paisley and Hostrup-Pedersen 2004) modelled the e ffect of level of impurities (0.02, 0.15, and 0.5 percent), inclusion of SRM, and other inputs on the probability of occurrence of BSE cases. Although the results were associated with a hi gh level of uncertainty, the study found that under certain scenarios tallow-based milk replacer coul d be associated with transmission of BSE to20 calves. The simulations demonstrated the importance of SRM exclusion in limiting the probability of BSE infection, part icularly from tallow with high impurity levels (0.5 percent). Uncertainty in the results stemmed from infectiv ity titer in CNS tissue and from the level of impurities in tallow.

Results of a quantitative risk assessment on the residual BSE risk posed by tallow (EFSA 2005)

concluded that tallow was not a risk factor in transmitting the BSE agent if the tallow was derived from cattle that have passed ante-mortem and post-mortem inspection. While the level of soluble impurities did not significantly affect the ri sk of exposure, they co ncluded that the source

of raw material warranted furt her consideration. In addition, re moval of SRM corresponded to a 2 log reduction in potential BSE infectivity. The EU Scientific Panel concluded that, even for the worst-case scenarios evaluated in the quantitative risk assessment, the exposure levels for tallow were minimal thereby posing no risk of transmission.

OIE guidelines. Import or transit of tallow w ith a maximum level of insoluble impurities of 0.15 percent by weight and derivatives made from th is tallow can be unrestricted regardless of BSE

risk status of th e cattle population.

Tallow with more than 0.15 percent insoluble im purities by weight requir es certification that:

 It is sourced from a ne gligible-risk country; or

 If it is sourced from a controlled-risk countr y, it is derived from cattle that have passed ante-mortem and post-mortem inspec tions and does not contain SRM.

Tallow derivatives

Supporting documentation. Several studies have evaluated the effect s of various time, temperature, and pressure proces ses on prion inactivation. As disc ussed in the previous section, the BSE-spiked tallow study (Taylo r et al. 1995) showed that while infectivity does not persist in tallow during rendering, the level of infectivit y of BSE subjected to a poorly inactivated rendering process remained at almost the same level as the untreated material.

Tallow derivatives are generally produced us ing manufacturing pro cesses that include hydrolysis, saponification, or tran sesterification under high temper ature and pressure conditions. Products made from this process have been show n to be safe regardless of the country from which they originate (title 21, Code of Federal Regulations, section 589.2001).

OIE guidelines. The import or tran sit of tallow derivatives othe r than those made from tallow

with a maximum level of inso luble impurities of 0.15 percent by weight requires certification

that:

 The tallow is sourced from a negligible-risk country; or

 If sourced from a controlled-risk country, the tallow is derived from cattle that have passed ante-mortem and post-mortem insp ections and does not contain SRM; or

 The tallow is produced by hydrolysis, saponi fication, or transester ification under high temperature and pressure conditions.

Dicalcium phosphate21 Supporting documentation. Considerable mineral cont ent is recovered from the hydrochloric acid treatment of bone chip used in the production of gelatin. As me ntioned earlier, before bones can be used in the manufacture of gelatin, fat a nd other impurities must be removed by degreasing, in

which the bones are crushed to a small size and then washed and degreased in a process that removes any residues of fat, marrow, or other so ft tissues. Before degreas ed bone chip material can be utilized to produce gelatin, minerals (i ncluding calcium and phosphate) must be removed. To remove minerals, the bone chip is soaked in hydrochloric acid (appr oximately 4 percent, pH <1.5) for a period of at least 2 days. The recovered minerals are further purified, followed by precipitation and drying. The resultan t product is dicalcium phosphate.

The same steps used in the pretreatment of bones to produce gelatin are followed for the production of dicalcium phosphate. Accordingly, studies that demonstrate the low BSE risk associated with gelatin resulting from the pr etreatment of bone dur ing degreasing and acid demineralization (Grobben et al. 2004; Manzke et al. 1996) also indicate that very low-risk dicalcium phosphate is yielded as a byproduct of the gelatin manu facturing process. Further, a significant reduction of TSE infectivity under experi mental conditions has been demonstrated for dicalcium phosphate by a recent validation study in which dicalcium phosphate was prepared

from bone artificially contaminated with TSEs (Grobben et al. 2006).

In addition, according to the EC SSC, a validatio n study by Grobben et al. shows that the acid process after degreasing and demi neralization as described in th e gelatin manufacturing process has a total clearance of infectivity of 2.6 l og10 (EC SSC 2003). The production process as a whole reduces the infectivity fu rther up to 3.8 to 3.9 log10, resulti ng in a residual proteinaceous fraction not exceeding 0.60 percent, of which 98 percent has a molecular weight below 10,000

Daltons.

The 2006 supporting document for the BSE Code Chapter 2.3.13 risk assessment (currently known as 11.6.28) results also points to the low risk of infectivity of dicalcium phosphate assuming different levels of contamination of bo nes with dorsal root gang lia and spinal cord, a very low infectivity of bone marrow, and an infectivity reduction for the acid process of 10 4.2

– 10 4.8 and for the heat/pressure process of 10 6.2 – 10 6.8 (OIE Terrestrial Animal Health Standards Commission [TAHSC] 2006).

In 2003, the EC SSC stated that the residual ri sk in dicalcium phosphate derived from bovine bones from was negligible when the raw material for the production of bovine bone dicalcium phosphate is (1) obtained from a country where BS E risk is considered negligible and derived from appropriate tissues (i.e., from animals fit for human consumption, with SRM [including skull and vertebrae] excluded and cross-cont amination with these bones avoided); or (2) submitted to a production process that has a proven TSE infectivity reduction capacity.

OIE guidelines. According to the OIE, dicalcium phosphate is not a risk factor for transmitting the BSE agent when it contains no traces of pr otein or fat. Dicalcium phosphate produced under

such conditions can be traded w ithout additional restrictions.22 However, if the dicalcium phosphate contains certain amounts of protein or fat, the OIE

recommends certain restrictions be applied for trade:

 It must originate from a country posing a negligible BSE risk; or

 It must originate from a country posing a c ontrolled BSE risk, it must be derived from cattle that have passed ante-mortem and pos t-mortem inspections, and SRM from cattle 30 months of age or older at the time of slaughter have been excluded.

Meat-and-bone meal

OIE guidelines. Ruminant meat-and-bone meal a nd greaves are considered to be one of the highest risk commodities for BSE. OIE recomm ends that ruminant meat-and-bone meal and greaves (or any products contai ning these commodities) can be traded only under the following

conditions:

 Products originate from a country cl assified as negligible risk; and

 Bovines from which the product originated were born after the date the ban was effectively enforced, if the exporting count ry has had an indigenous case of BSE.

Skeletal muscle meat

This section discusses potential BSE infectivity in skeletal muscle meat from several sources: the meat itself, blood, and cross-contamination.

Supporting documentation. BSE infec tivity has not been demonstrated in the muscle tissue of

BSE-infected cattle examined in either the m ouse bioassay or in ca ttle assays in the UK pathogenesis study (Wells 1996, 2005, 2008). Some reports have detected the presence of prions in muscle tissue from rodents, humans, and sma ll ruminants infected with TSEs other than BSE (Bosque 2002). Those findings are consistent with differences in the transmission, host range, genetic susceptibility, in fectivity distribution, and epidemio logy found in different TSEs that affect animals and humans. However, in transg enic mice that overexpress the bovine PrP gene (Tg bov XV), infectivity was detected in one mu scle (semitendinosus) from a single clinical case of BSE in Germany (Buschmann and Groschup 2005) . Most recently (Balkena-Buschman et al, 2011) using transgenic mice overexpressing the bovine prion protein challenged with a variety of tissues collected from terminally ill cattle repor ted the presence of BSE infectivity in tongue in

absence of detectable PrPsc accumulation. The sensitivity of these mice to infection is significantly greater than that of the mice used for the UK pathogenesis study (10,000-fold) and even greater than that of cattle (approximately ten-fold).

From the UK studies of the pathogenesis of expe rimental BSE in cattle, no infectivity is found in assays of skeletal muscle pools (triceps, ma sseter, sternocephalicus, and longissimus dorsi) completed in wild-type mice bioassa y and in cattle bioassay of sk eletal muscle pools (masseter, semitendin-osus, and longissimus dorsi) from select ed kill time points of the oral exposure study (Wells et al. 1996; Wells 2005). All assays of the skeletal muscle pools were completed in March 2007 (Wells 2008).23 Recent studies using tissues from asymptomatic catt le challenged orally with BSE; culled at 20, 24, 27, 30, and 33 months; and inoculated intracereb rally into BoPrP-Tg110 mice have failed to detect infectivity in muscle (Espinosa 2007).

The international scientific community largely co nsiders that bovine muscle tissue from animals

infected with BSE does not contain detectable amounts of BSE infectivity. For example, the UK’s Spongiform Encephalopathy Advisory Committee (SEAC 2001) and the EC SSC evaluated the implications of the findings of the presence of infec tivity in muscle for other TSEs in different species in relation to human food sa fety. EC SSC concluded that there was no reason to revise its opinions regarding the safety of meat, given the c onsistent negative results in BSE infectivity experiments (EC SSC 2002d). SEAC concl uded that the findings could not be directly applied to BSE in cattle and did not change the assessment of the risk to humans of consumption of beef. Updated EC SSC opinions remain consistent with their original reports.

With the additional safeguards of recommende d stunning practices (EFSA 2004; International

Forum for Transmissible Animal Diseases and Food Safety 2009) and th e hygienic removal of SRM (EFSA 2005), skeletal muscle meat itself is considered safe.

In regard to the infectivity of skeletal meat from blood, experi mental evidence indicates that

blood does not constitute a risk of BSE transm ission (see section on blood and blood products).

Any BSE risk in deboned skeletal muscle meat is derived primarily from the potential for crosscontamination from BSE-infected materials during slaughter and/or processing. However,

controls can be established to ensure that co ntamination of the beef with the BSE agent is unlikely to occur. These cont rols are directed toward pr ocedures for stunning, pithing, 4 removal of SRM, and carcass splitting. For instance, several studies have shown that penetrative captive bolt stunners that incorporate air-injection can fo rce visible pieces of br ain and other CNS tissue into the circulatory system of stunned cattle. In addition, the pithing process could cause

dissemination of central nervous tissue th roughout the body. Avoidi ng these procedures eliminates the potential of this risk. Finall y, SRM must be handled in ways that prevent contamination of the carcass since, their remova l is the most critical mitigation preventing contamination of edible meat with BSE infectivity.

OIE guidelines. Experimental evidence indicates that deboned skeletal muscle meat is not a risk factor in transmitting the BSE agent if the appropriate mitigations measures are applied to prevent contamination with SRM. Therefore, OI E guidelines recommend that regardless of the BSE risk status of the country, the trade of deboned skeletal muscle meat from cattle is considered safe provided that:

a. Cattle were not subjected to a stunning pr ocess prior to slaughter with a device injecting compressed air or gas into the cranial cavity or to a pithing process;

b. Cattle passed ante-mortem and post-mortem inspections; and

c. The meat has been prepared in a manner to avoid contamination with SRM tissues.

4 Pithing involves the insertion of an elongate d rod-shaped instrument into the cranial cavity of a stunned animal to further lacerate the central nervous system tissue.24 Meat products and byproducts

This section outlines the OIE criteria for meat pr oducts, which are the muscle and parts that can

include but are not limited to cert ain organs of a slaughtered anim al that can be for both human

and animal use.

OIE guidelines. Requirements for trade in meat and meat byproducts are based on the status of the exporting country. For safe exports of meat products and byproducts, the veterinary authority must make specific certifications based on the BSE status of the exporting country.

For countries classified as negligible risk:

a. Cattle passed ante-mortem and post-mortem inspections; and

b. In the event the country had reported indige nous cases of BSE, the cattle from which the fresh meat and meat products were deri ved were born after th e date the ban on the feeding of ruminants with meat-and-bone m eal and greaves derived from ruminants has been effectively enforced.

For countries classified as controlled BSE risk:

a. Cattle were not subjected to a stunning pr ocess prior to slaughter with a device injecting compressed air or gas into the cranial cavity or to a pithing process;

b. Cattle passed ante-mortem and post-mortem inspections;

c. Fresh meat and meat products do not contai n mechanically separated meat from the skull and vertebrate column of cattle 30 months of age or older; and

d. SRM have been removed in a manner that avoids contamination as defined for

controlled-risk countri es (Article 11.6.14).

For countries classified as undetermined BSE risk:

a. Cattle from which the fresh meat originates were not fed meat-and-bone meal or greaves derived from ruminants;

b. Cattle were not subjected to a stunning pr ocess prior to slaughter with a device injecting compressed air or gas into the cranial cavity or to a pithing process;

c. Cattle passed ante-mortem and post-mortem inspections;

d. Fresh meat and meat products do not contai n mechanically separated meat from the skull and vertebrate column of cattle over 12 month of age and older; and

e. SRM (as defined for undetermined-risk countries) have been removed in a manner

that avoids contamination with SRM and w ith nervous and lymphatic tissues exposed during the deboning process.

Blood and blood products

Blood and blood products can be divided into two main groups: whol e blood and cellular derivatives such as red cell concentrate, platelet s and other cellular elements; and plasma-derived products including serum (including fetal bovine serum, clotting factors, immunoglobulins, and albumin). Plasma is the cell-free portion of th e blood. Serum is plasma with fibrinogen and clotting factors removed.25 Supporting documentation. BSE infectivity was not de monstrated in cattle blood or any tested derivatives (EC SSC 2000). This conclusion derives from studies in which tissues from infected cattle were injected intracerebrally and intr aperitoneally into mice (mouse bioassay), or intracerebrally into cattle (cattle bioassay). Mouse bioassays were performed using buffy coat (the white cell fraction of centrifuged whole blood), clotted blood, fe tal calf blood, and serum from confirmed clinical cases (EC SSC 2000). Wild-type mouse and cattle bioassays were performed on buffy coat from cattle experimentally exposed orally to the BSE agent. In all cases, no evidence of infectivity was detected. Howeve r, brain damage caused by certain stunning techniques can produce CNS tissue emboli in venous blood draining the head (EFSA 2004). A study (Espinosa et al. 2007) utiliz ing material derived from the second Veterinary Laboratories Agency UK pathogenesis study (cattle challenged orally with BSE and culled at 20, 24, 47, and 30 months post exposure) revealed no detectable blood infectivity by assay in transgenic BoPrPTg110 mice.

Investigators have demonstrated that BSE can be transmitted to sheep by transfusion of whole blood from sheep experimentally infected with BSE (Houston et al. 2000; Hunter et al. 2002). In these studies, a transfusion of 400 ml of whole blood, taken fro m clinically normal infected sheep, caused disease in 2 of 24 r ecipients. Blood or buffy coat take n from clinically ill animals, however, did not cause disease in the four recipients. These same investigators also examined scrapie in sheep. A total of 4 sh eep out of 21 transfused with bl ood from sheep naturally infected with scrapie developed disease. The transfusion of buffy coat derived from a clinically ill animal caused disease in the recipient. The EC SSC exam ined these studies and their implications and concluded that the finding of in fectivity in the blood of sheep c ould not be extrapolated to BSE in cattle (EC SSC 2002c).

Brown et al. (1999), using a human strain of TS E in mice inoculated in tracerebrally, concluded that infectivity was present in the buffy coat durin g the preclinical phase of TSE but absent or in only trace amounts in the plasma or plasma fractions. Following the onset of clinical signs, increased infectivity of both buffy coat and pl asma was found, but sti ll at very low levels compared to the CNS. As cited in a review of the relevant literature (Comer 2004), most studies using a rodent model and adapted strains of sc rapie or CJD demonstrat ed that the fractions containing white blood cells have th e highest levels of infectivity.

In contrast to investigations of the natural di stribution of infectivity in rodent blood fractions, one “spiking” study added high levels of hams ter-adapted scrapie infectivity from brain homogenate to normal human blood. Following fr actionation by centrifugation into red cells, white cells/platelets, and plasma components, titra tions indicated that the majority of infectivity was in the red cell component (Brown et al. 1998 ). These results, although not as relevant to understanding the natural distribu tion of TSEs in blood, may potenti ally apply to the distribution following cross-contamination at bl ood collection. Therefore, if any BSE infectivity is present in bovine blood, either naturally or via cross-c ontamination, it would likel y be highest in the cellular components. These fract ions, both red and white cells, are excluded when harvesting

fetal bovine serum and bovine serum albumin us ed in the preparation of vaccines and drugs.

Further decrease in TSE infectivity occurs with fractionation of plasma pr oteins. Fractionation is the process whereby specific proteins, such as albumin, are separated from other components of26 the plasma. Infectivity in various fractions has been examined. For example, using data from
several cited studies, Comer (2004) estim ated that human albumin contains 3.1  10 -5 vCJD

ID 50 /gram. Compared to Comer’s estimates of infectivity in whole blood (2 iv vCJD ID 50 /gram), this figure represents a dramatic decrease.

The possibility of cross-contamination of bl ood products with SRM exists at the time of collection, particularly in a slaughter environment. Certai n slaughterhouse stunning practices

 specifically the use of de vices that inject compressed air or ga s into the cranial cavity or pithing

processes

 may introduce macroemboli of CNS tissue into the circulatory system (Anil et al. 1999; Schmidt et al. 1999). In addition, collectio n of blood in an open manner may allow other

tissues to contaminate the blood.

OIE guidelines. To prevent contamination due to such potential source s of infectivity, the following mitigations are required:

For all blood:

1. The blood is collected in a manner that avoids contamination.

For blood collected at slaught er, the slaughtered animal:

1. Passed ante-mortem inspection; and

2. Was not subjected to a stunning process with a device injecting compressed air or gas into the cranial cavity or to a pithing process.

Live cattle

Supporting documentation. The primary source and onl y documented route of field transmission

of BSE infection is commercial feed contaminated with ruminant protein derived from infected animals (Wilesmith et al. 1988, 1991, 1992; Prince et al. 2003). Feed bans that prohibit incorporation of ruminant protein into ruminant feed have shown to be effective in mitigating the risk of BSE transmission. The UK experience dem onstrated the effectiveness of implementing a ruminant-to-ruminant feed ban on signifi cantly reducing the prevalence of BSE.

The 165 cases of BSE born in the UK after the imp lementation of the feed ban suggest possible exposure to an exogenous feed source from continental Europe, which still permitted legal trading of meat-and-bone meal until January 200 1. Cross-contamination of feedstuffs and ingredients has also occurred in a number of other BSE-affected countries (Hill 2005).

OIE guidelines. Trade of live bovines from negligib le and controlled BSE risk countries should be allowed under the following conditions:

a. For negligible BSE risk: Animals are identifi ed by a permanent identification system in such a way that demonstrates that they were not exposed to BSE cases or their cohorts, and, if the country has had an indigenous cas e of BSE, the animals were born after the date of the ban on using meat-and-bone meal and greaves of ruminant origin was effectively enforced.

b. For controlled BSE risk: The country comp lies with the conditi ons under Article 11.6.4, animals are identified by a permanent iden tification system in such a way that27 demonstrates that they were not exposed to BSE cases or their cohorts, and they were

born after the date of the ban on using meat-and-bone meal and greaves of ruminant origin was effectively enforced.

The OIE recommends that undetermined-risk countr ies can export live cattle if the country has an effectively enforced ruminant -to-ruminant feed ban and the cat tle selected for export were born 2 years after the date the ban was eff ectively enforced. Trade of live bovines from

undetermined-risk countries shou ld be allowed provided that:

a. The country has adequate infrastructure and capabilities to establish and enforce the feed ban;

b. The ban is effectively enforced; and

c. The cattle selected for export:

1. Were born at least 2 years after the date of the ban on using meat-and-bone meal and greaves of ruminant origin wa s effectively enforced; and

2. Are identified by a permanent identification system in such a way that demonstrates that the cattle were not exposed to BSE cases or their cohorts.

However, APHIS believes that verifying that th ese conditions have been met would be difficult for undetermined-risk countries. In considering the overall BSE risk of an exporting country,

factors in addition to a feed ba n — such as veterinary infrastr ucture, surveillance, and import controls — play a role. Such f actors can be taken into consid eration if a country has been categorized for BSE risk. When an undetermined-ri sk country demonstrates that an effectively enforced feed ban is in place and confirms th at the other conditions have been met (both necessary for exporting live bovines), APHIS believe s the region would most likely qualify as at

least controlled risk.

A second reason for not allowing the importation of live bovines from undetermined-risk regions is based on practical constraints. Considering the conditions for the export of meat, meat food products, or meat byproducts (other than boneless skeletal muscle meat) from undetermined-risk countries, SRM would have to be removed from bovi nes over 12 months of age that originated in undetermined-risk countries and are slaughtered in the United States. Ensuring the removal of SRM from these animals would be difficult, n ecessitating special identification and tracking

while in the United States and at slaughter by both APHIS and FSIS, which oversees the removal of SRM. Therefore, the importa tion of live bovines from countries of undetermined risk for BSE should be prohibited except in very limited situations.

Live animals for specific uses such as exhib itions, zoos, etc., should be allowed only on a caseby-case basis.

Animal identification system

Articles 11.6.7 to 11.6.9. of the OIE Code recommend that cattle for export be identified by a permanent identification system to enable the expor ting country to show that they have not been exposed to any BSE cases (i.e., are not exposed cattle). Exposed cat tle are described as:

 An individual animal infected by the BSE agent, with or without clinical signs;28  Cattle that, during the first year of life, were reared with BSE-affected cattle during the first year of the BSE-affected cattle’s life and that, per investigation, consumed the same potentially contaminated feed during that period; or

 Cattle born in the same herd as the BSE-affected cattle, within 12 months of the birth of

the affected cattle if the feed investigation was inconclusive.

For negligible and controlled-risk countries, the internati onal veterinary certif icate must describe the permanent identification system that demons trates that the cattle to be exported are not exposed cattle.29 

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Wrathall, A. K. Brown, A. Sayers, G.A.H. Wells, M. Simmons, S. Farrelly, P. Bellerby, J. Squirrell, Y. Spencer, M. Wells, M. Stack, B. Bastiman, D. Pullar, J. Scatcherd, L. Heasman, J. Parker, D.A.R. Hannam, D. Helliwell, A. Chree, and H. Fraser. 2002. Studies of embryo transfer from cattle clinically affected by bovine spongiform encephal opathy (BSE). Vet. Rec. 150(12): 365-378.

Yamakawa, Y., K. Hagiwara, K. Noht omi, Y. Nakamura, M. Nishijima, Y. Higuchi, Y. Sato, T. Sata, and the Expert Committee for BSE Diagnos is, Ministry of Health, Labour and Welfare of Japan. 2003. Atypical proteinase K -resistant prion protein (PrPres) observed in an apparently healthy 23-month-old Holstein st eer. Jpn. J. Infect. Dis. 56: 221-222.35 

### Appendix: Characteristics of the BSE Agent

This appendix describes the characteristics of the BSE agent, including transmission, incubation period, tissue distribu tion, and infectivity in cattle, its most common host.

Transmission

Oral ingestion of feed contaminated with the abnormal BSE prion protein is the only documented route of field transmission of BSE (Prin ce et al. 2003; Wilesmith et al. 1988, 1991, 1992). Contamination occurs when abnormal proteins de rived from specific tissues originating from infected ruminants are incorporated into feed. Standard rendering pro cesses do not completely inactivate the BSE agent. Theref ore, rendered protein such as meat-and-bone meal derived from infected cattle may contain the infectious agent. Bans prohibiting incorporation of ruminant protein into ruminant feed are imposed to m itigate the risk of BSE transmission (OIE, 2009).

Early studies suggested that maternal transmissi on of BSE is possible. A cohort study conducted in the UK indicated that offspring of BSE-affected dams were at a higher risk of also developing clinical BSE (Wilesmith et al. 1997). These results were consistent with a rate of maternal risk enhancement of approximately 10 percent in the offspring born within 12 months of the dams’ onset of clinical signs of BSE. Since the initial cohort study, ho wever, modelling studies based on the epidemic data from the UK have indicated that the cumulative maternal risk is only 1 percent in calves born in the la st 6 months of incubation of th e disease in its dam (Donnelly et al. 2002). More recent work on cases born after th e 1996 feed ban fails to demonstrate evidence of maternal transmission (Hill 2005). Thus, alt hough maternal transmission may be possible, more recent epidemiologic evidence suggests that maternal transmission of BSE is unlikely to occur at any appreciable level, if at all.

Epidemiological studies and mathematical modell ing have supported the view that most cases were likely exposed as calves (Wilesmith et. al 1988) with the greate st chance of becoming infected up to or around the firs t year of life (Wilesmith et al. 1992, 1992a; Ferguson 1997; De Koeijer et al. 2004; Arnold and Wilesmith 2004). Th ese findings suggest that susceptibility in cattle declines with age, and therefore young animals are most susceptible. One mathematical model assumes that susceptibility declines expone ntially after the age of 4 months, levelling off at 10 percent of the peak va lue (De Koeijer et al. 2004).

A study from Great Britain published the same year (Arnold and Wilesmith 2004) used unpublished data on the feeding of proprietary concentrates to re placement dairy cattle and BSEepidemic data to back-calculate the likely age of infection. This study found that dairy cattle were at greatest risk in thei r first 6 months. Although this resu lt likely confounds susceptibility with exposure because of age-dependent patterns of nutritional supplementation, the two characteristics are effectively linked.Appendix: Characteristics of the BSE Agent

36 Experience in the UK demonstrates that implem entation of a ruminant-to-ruminant feed ban exerts downward pressure on the prevalence of BS E (Figure 1). Animal feed restrictions began in the UK in July 1988, when the use of ruminant meat-and-bone meal in ruminant animal feed was banned. In September 1990, the use of specifi ed bovine offals was banned for use in any animal feed in the UK. This ban prohibited the us e in any animal feed of bovine tissues with the highest potential concentra tion of infectivity. In 1994, the use of mammalian protein

 not just ruminant protein

 was banned from ruminant feed. In 1996, feeding of any farmed livestock, including fish and horses, with mammalian m eat-and-bone meal was completely banned. As a result of these bans to reduce the recycling of infectivity, the annual incidence of BSE fell by 99.9 percent, from 36,682 in 1992 to 48 in 2007 (D epartment of Environment, Food, and Rural Affairs [DEFRA] 2008).

When the UK epidemic is plotted by year of birth, the impact of the feed ban is striking. Although the data that are presen ted in the following figure and table represent the specific

situation in the UK 1 from 1974 to 2002, we expect similar effects in any country that implements a comparable feed ban.

Figure 1. Effect of feed ban on BSE cases in Great Britain. The first feed ban was implemented in the summer of 1988 before fall calving (DEFRA 2008).

The raw data that provided the basis fo r figure 1 are reproduced in table 1.

1 The data made available by DEFRA on its Web site are for Great Britain and, in some cases, for Northern Ireland. These data

exclude the Channel Islands and Isle of Ma n. Records published elsewhere indicate that the vast majority of BSE cases in the UK have been detected in Great Britain (OIE 2008). Therefore, ep idemiologic patterns noted in Great Britain are considered to represent the overall UK experience.Number of BSE cases



### Year of birth of BSE cases Feed Ban ImplementationAppendix: Characteristics of the BSE Agent

37 Table 1. Confirmed cases in Great Britain by year of birth, where known

Year Cases Year Cases
1974 1 1989 12,748
1975 0 1990 5,750
1976 2 1991 4,781
1977 10 1992 3,539
1978 6 1993 3,010
1979 41 1994 2,196
1980 102 1995 1,112

1981 262 1996 70
1982 1,394 1997 45
1983 4,463 1998 43
1984 8,069 1999 27
1985 11,071 2000 8
1986 19,754 2001 6
1987 36,938 2002 4

1988 22,268 Unknown birth

year 43,344

Total cases: 181,064

Source: DEFRA 2008.

Despite the dramatic evidence presented above , approximately 150 cases of BSE have been

detected in animals in the UK born after the ba n was reinforced in 1996 (BAB cases). Several analyses of the BAB cases suggest that the cat tle were likely to have been exposed to an exogenous feed source from continental Europe. Fo r instance, an analysis of the first 16 cases (Wilesmith 2002) indicates that an exogenous (n on-UK) feedborne source remains the most likely explanation, since trading of meat-and- bone meal was legally possible at ports of Continental Europe until the European-wide ba n was introduced in January 2001. Significant amounts of ingredients for cattle feed were imported into the UK before 2001, and crosscontamination of feedstuffs and ingredients ha s occurred in a number of other BSE-affected countries. A recent report (Hill 2005) also descri bes the association of BAB cases with imported feed until controls were strengthened throughout Europe.

In 2007, the EU reported that, of the cattle born after 2000 that tested positive for BSE, only 4 cattle were less than 40 months of age (UK, 36 and 39 months; Portugal, 32 months; Poland, 25 months) with some uncertainty about the age of the Polish case. To date, the majority of BAB cases (18/22) were born in 2001. So far, three ca ses have been born in 2002 (UK, Portugal and Spain) and one in 2003 (Poland), which tested po sitive at only 25 months of age (EFSA 2007).

Incubation

BSE has a prolonged incubation period. Epidemio logical data from the UK epidemic have demonstrated that, on average, cattle develop clin ical signs 4 to 6 years after infection (Bradley 1991; Anderson et al. 1996). In BSE, as in othe r TSEs, the total amount of infectivity in anAppendix: Characteristics of the BSE Agent

38 animal increases throughout the incubation period reaching the highest load at the end, very

close to the death of the anim al. Infectivity is considered to increase exponentially after exposure, reaching 3 logs less than clinical cas es by 70 percent of the incubation period, and 4.5 logs less than a clinical case at 50 percent of the incubation period (Comer and Huntly 2003).

The incubation period is thought to be inversely related to dose (i.e., low-dose exposures have long incubation periods before clin ical signs of disease become apparent). The Department for Environment Food and Rural Affairs (DEFRA) UK ha s carried out two oral challenge studies in cattle to determine the incubation period, inoculat ing varying amounts of brainstems pooled from BSE-affected cattle (Wells et al. 2007). In the fi rst attack rate experiments, groups of 10 calves were dosed orally with 3  100 g (100 g on 3 successive days), 100 g, 10 g, or 1 g of brain tissue from clinically sick cattle. All anim als in the two higher dose categories (3  100 and 100 g, respectively), 7 out of 9 in the 10-g trial group and 7 out of 10 in the 1-g group developed clinical BSE. The incubation period (IP) for the 3  100 g ranged between 33 and 42 months. The IP for the 100 g was 33 to 61 months; for the 10 g was 42 to 75 months; and for the 1 g was 45 to 75 months. The remaining animals in this experiment were killed at 110 months after exposure and showed no patholog ical evidence of disease.

The second attack rate experime nts extended these findings with lower doses (1 g, 0.1 g, 0.01 g, and 0.001 g). Wells et al. reported 3 of 4 animals as positive for BSE in the 1-g trial group (IP range 58–73 months), 7 out of 15 animals in th e 0.1-g group (IP range 53–98 months), 1 out of 15 in the 0.01-g group (IP range 56 months), an d 1 out of 15 in the 0.001-g group (IP range 68 months). Combined results of the 1-g dose groups from the phase 1 and 2 studies showed 10 out of 14 as positive (IP range 45–73 months). The study concluded that the estimated dose at which 50 percent of the cattle will be clinically affected was 0.2 g of infected brain.

According to DEFRA, the dose between 1 and 10 g most closely fits the picture of incubation periods seen in the epidemic. The shortest in cubation period for 1 g dose is 45 months and the mean is 60 months, which suggests that the infec tivity in CNS-related SRM would most likely be absent in cattle aged 35 months.

The UK data show that of the cattle that develo ped clinical BSE in the field, only 0.01 percent were less than 30 months of age (EC SSC 2002). T hus, cattle less than 30 m onths of age are less likely to be in the later stages of BSE incuba tion than older BSE-infected cattle and hence are less likely to contain high levels of BSE infec tivity. In addition, research demonstrates that the incubation period for BSE appears to be linked to the in fectious dose of the BSE agent received, i.e., the larger the infectious dose received, the shorter th e incubation period (EC SSC 2002). Given these observations, scientists believe that the occurrence of BSE in young cattle is most likely the result of exposure to a very large dose of the BS E agent at a very young age.

Tissue distribution and infectivity

Most of the information on the development and di stribution of tissue infectivity in BSE-infected cattle has been derived from experimental pathog enesis studies conducted in the UK (Wells et al. 1994, 1998, 1999). In these studies, cattle were de liberately infected with BSE through oral exposure to the brain tissue of ca ttle with confirmed BSE. Subsets of the experimentally infectedAppendix: Characteristics of the BSE Agent

39 cattle were killed at regular intervals as the dis ease progressed. At each interval, the tissues of the infected cattle were examined for histopath ological changes consistent with BSE and for abnormal prion proteins. Also, at e ach interval, a mouse assay was done

 tissues of the BSE infected cattle were injected intracerebrally and intraperitoneally into mice to identify those

tissues of cattle c ontaining infectivity.

The UK pathogenesis studies involved 30 animals, each of which received a single dose of 100 g of infected brain at 4 months of age (We lls et al. 1994, 1998, 1999). This dose is probably 10 to 100 times greater than that associated w ith field exposure via f eed (DEFRA 2006). The

studies demonstrate that in cattle infected with BSE, the total amount of infectivity in the animal,

as well as the distribu tion of infectivity in the animal’s body, changes over time (Wells et al. 1994, 1998, 1999). First, infectiv ity was detected in the distal ileum of cattle from 6 to 18 months after oral exposure. The highest levels of infectivity were detected in the brain and spinal cord at the end stages of disease. Some cattle exhibited clinical signs of BSE as early as 35 months post oral exposure to the BSE agent. By 37 months post oral exposure, all five animals

that were still alive demonstrated clinical evidence of BSE. 2 Infectivity was f ound in cattle with clinical signs of BSE in the br ain, spinal cord, dorsal root ga nglia, trigeminal ganglia, and the

distal ileum of the small intestine.

A different study (Espinosa et al. 2007), utiliz ing material derived fr om the UK pathogenesis study (100-g dose of BSE-infected br ainstem materials orally inocul ated into cattle), reported the detection of infectivity (by a ssay in transgenic BoPrP-Tg110 mi ce genetically altered to be highly susceptible to BSE) in the brainstem of ca ttle killed 27 months af ter exposure. A different study (Masujin et al. 2007) detect ed the presence of PrPsc in th e vagus nerve and adrenal gland obtained from clinically confirmed BSE cases

BSE infectivity was demonstrated in the brain, spin al cord, and dorsal root ganglia as early as 32 months after oral exposure to the BSE agen t in some cattle (Wells et al. 1994, 1998, 1999). Infectivity was demonstrated in these tissues 3 m onths before animals began to develop clinical signs of the disease. In addition, infectivity was demonstrated in the distal ileum of cattle 6 to 18 months after oral exposure to the BSE agent a nd again at 38 months a nd 40 months after oral exposure. Regarding the distal ileum, st udies examining the distribution of PrP Sc by immunohistochemistry of experimentally infected cattle confirmed that PrP Sc accumulated in Peyer’s patches of the distal ileum at 6, 10, 14, 18, 36, and 40 months after exposure (Terry et al. 2003). The same study failed to detect PrP Sc by immunostaining in the duodenum, jejunum, cecum, colon, myenteric plexus, and submucosal pl exus of calves orally exposed to large doses of the BSE agent and killed 6 months after expos ure. In contrast, immunostaining of follicles from Peyer’s patches of the distal ileum did reveal the presence of PrP Sc in these same calves. The distal ilea of 29 naturally occurring cases of BSE were examined for PrP by immunohistochemistry. Sparse PrP Sc -specific immunostaining was observed in neurons of the

2 CNS tissue pools of three animals sacrificed at 22 months and one animal sacrificed at 26 months post cha llenge did not contain infectivity when bioassayed in cattle (Wells et al. 2005).Appendix: Characteristics of the BSE Agent

40 myenteric plexus of the distal ileum of nine of the cattle. No immunostaini ng in the submucosal plexus of the distal ileum of a ny of the infected cattle was obse rved, suggesting a very low level of infectivity in the en teric nervous system in the clinical phase of BSE (Terry et al. 2003). A recent study of intestinal samples obtained from the German BSE pathogenesis study (Hoffman et al, 2011) found high amounts of infectivity in th e distal ileum, moderate amount in ileocaecal junction and traces of infectivity in the jejunum. The higher amount s of infectivity were mainly seen in younger cattle (8 to 12 months post inoc ulation). Recent examination of intestinal samples obtained from the UK pathogenesis stud y (Stark, 2011) using the mouse bioassay found immunostaining of the intes tinal nervous system was rare . Since the enteric nervous system extends throughout the small intestine, th e recent findings indicate that infectivity in animals exposed to high doses of the agent could extend to the jejunum and the duodenum.

A second phase of the UK pathogenesis studies, wh ich used a cattle bioassay as an endpoint, was

conducted to ensure that low levels of infectivity that may not ha ve been detected in the first phase using the mouse bioassay were not misse d (DEFRA 2006; EC SSC 2002). This second phase of the study was completed in March 2007 (Wells 2008). In the cattle bioassay, tissues from the same cattle orally exposed to BSE in the earlier pathogenesis studies were injected directly into the brain of BSE- free cattle (DEFRA 2006). This method is considered to be several

hundred-fold more sensitive in detecting BSE infectivity than the standard mouse bioassay (DEFRA 2006). Results from the cattle bioassa y study demonstrate that, in addition to the materials that were found to contain infectivity when the mouse bioassay was used, the tonsils of calves 10 months post oral exposure to the BSE agen t also contain infectivity (Wells et al. 2005). However, because only one of five animals injected with tonsil material from infected animals developed clinical BSE at 45 months post-inocul ation, the level of inf ectivity in the tonsils appears to be very low.

A larger pathogenesis study involved 56 calves that were orally challenged with macerated brainstems from 225 BSE-positive cattle (EFSA 2007; Hoffman 2007). The infectivity in the BSE brain stem homogenate used for the cattle infection study was 106.1 ID 50 per gram of tissue as determined by endpoint titration in T gbov XV mice (Buschmann and Groschup 2005). Every 4 months, four or five randomly selected animals are euthanized and necropsied, and more than 150 tissue and bodily fluid samples are collected from each animal and analyzed by immunohistochemistry, pure-tone average West ern blot, and transgenic mouse bioassay (TgbovXV). The aim of the study was to allow a better understanding of the pathogenesis and to

determine the earliest time of detection of BSE in the CNS. The initial results from the German BSE pathogenesis study demonstrate that BSE prions can reach the brain as soon as 24 months after a massive oral challenge (Hoffman 2007). A recent study of intestinal samples obtained from the German BSE pathogenesis study (H offman et al, 2011) found high amounts of infectivity in the distal ileum, moderate amount in ileocaecal junction, and tr aces of infectivity in the jejunum.

The amount and distribution of inf ectivity in specific tissues from an infected cow have been estimated by Comer and Huntley (2003) in their eval uation of the availabl e literature. Those summary results, presented in table 2, describe distribution of infectiv ity in various tissues, including brain, spinal cord, dorsa l root ganglia, trigeminal ganglia , tonsil, and distal ileum of a

BSE-infected cow. The table uses an estimated weight of each tissue in grams, the number ofAppendix: Characteristics of the BSE Agent

41 estimated cattle oral infectious dose-50 (ID 50 ) units 3 per gram, and the total number of cattle oral

ID 50 units attributed to each tissue to es timate a percentage of cattle oral ID 50 units for each

tissue.

3 BSE infectivity is expressed in terms of cattle oral infec tious dose-50 units (ID 50 ). A cattle ID 50 is defined as the amount of infectivity required to cause infection in 50 percen t of an exposed cattle population (Cohen et al. 2001).Appendix: Characteristics of the BSE Agent

42 Table 2. Infectivity in a clinical case of BSE (cattle oral ID 50 )

Tissue Weight (g/animal) Infectivity % ID 50 /g ID 50 /animal
Brain 500 50 25,000 60.2 Spinal cord 200 50 10,000 24.1 Dorsal root ganglia 30 50 1,500 3.6 Trigeminal ganglia 20 50 1,000 2.4

Tonsil 50 0.005 0.25 0.0
Distal ileum 800 5 4,000 9.6

TOTAL 1,600 41,500

Source: Comer and Huntly 2003

The table shows that over 90 percent of the infectivity in clinica lly affected cattle is associated with central and peripheral ner vous system tissues, i.e., brain, spinal cord, dorsal root ganglia, and trigeminal ganglia. About 10 percent was as sociated with the di stal ileum. Minimal infectivity was associated with tonsil s in a clinically affected animal.

Bones

Early studies of cattl e challenged orally with 100 g of BSE in fected brain tissue (Wells et al. 1996, 1998) and analyzed at approximately 4-month intervals from 2 to 40 months post exposure showed that infectivity was dete cted, using the mouse bioassay, in the sternal bone marrow from pooled samples of animals killed in the clin ical phase of the disease at 38 months post

inoculation but not before a nd not after (Wells et al. 1999). Infectivity was detected in the bone marrow from the cattle (n=3) killed at 38 months but not those killed at 32, 36, or 40 months. Of the 16 mice inoculated with the 38-month sample, 2 showed clinical sign s of disease at 695 and 842 days and were positive for BSE. In addition, four other mice were positive by PrP immunohistochemistry alone (with survival periods of 653–923 days). The inconsistent results of

the absence of detectable infectivity in bone marrow in this study at the later time point of 40 months post inoculation has raised the possibility that the finding of infectivity at 38 months post inoculation may have been the result of an accidental procedural contamination.

To improve the sensitivit y of the oral exposure study, additional assays of selected tissues were inoculated intracerebrally in cattle. These studie s are approximately 500-fold more sensitive than the standard mouse bioassay. Bone marrow sample s taken at 32 and 36 months (plus those taken at 22 and 26 months) after oral exposure were each inoculated intracerebrally into five 4-monthold cattle. Results of the studies did not replicate the presence of infectivity in bone marrow (Wells 2008). In addition, results from a la rger pathogenesis study in Germany did not

demonstrate PrP sc in the bone marrow (femur) of two clin ically normal cows killed after 24 and 28 months post exposure.Appendix: Characteristics of the BSE Agent

43 Studies in transgenic mice

In the last few years, BSE research has incorp orated the use of transgenic mice designed to overexpress bovine prion protein as a way to im prove the sensitivity and the efficiency of

transmission studies.

In a recent study, mice genetically engineered to be highly susceptible to BSE and to overexpress the bovine prion protein (Tgbov XV mice) were inoculated with tissues from an end-stage clinically affected BSE-infected cow (Buschma nn and Groschup 2005). The sensitivity of these mice to infection was shown to be significantly great er than other mice panels used in bioassays. In addition, the sensitivity was s hown to be even greater than that of cattle by approximately tenfold. This study demonstrated low levels of inf ectivity in the facial a nd sciatic nerves of the peripheral nervous system when injected into these highly sensitive mice. A more recent study (Espinosa et al. 2007), which also used highly sensitive genetically engineered mice but inoculated with tissues from asymptomatic cattle , also detected low levels of infectivity in bovine sciatic nerve. In another study, PrPsc was al so detected in the vagus nerve, adrenal gland, and weakly from the sciatic nerve from clinic ally confirmed BSE cases (Masujin 2007).

While these are findings that can help further ch aracterize the pathogenesi s of BSE, they cannot be easily extrapolated into the context of the ri sk presented by natural exposure pathways. The findings may be influenced by the overexpressi on of prion proteins in these genetically engineered mice. Any apparent levels of infec tivity are low and would be even lower in other species such as cattle. Given all of these factor s, we conclude that there is not sufficient information in this study to alter our understandi ng of the epidemiologically significant distribution of BSE infectivity in cattle.

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