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 Criteria 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 restricts the importation of live ruminants and ruminant products 

from certain countries1

 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 regulations (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 OIE’s Terrestrial Animal Health Code (the Code) 

Chapter 11.6 (OIE 2009) by (1) adopting the categories and criteria for classification 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 for BSE risk by the OIE, APHIS is 

proposing to base import policies on that classification. For a country that is not yet classified by 

the OIE as to BSE risk, APHIS would, upon request 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-infected imports. Proposed changes would be 

consistent with the most current scientific understanding of BSE risk, based on the 

characteristics of the disease (including transmission) and risk mitigations necessary to protect 

animal health. 

This supporting scientific documentation summarizes the significant components of the relevant 

OIE BSE Chapter, including (1) the three levels of risk status classification 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) recommendations for restricted or unrestricted trade 

in certain commodities according to the BSE 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 regulations date back to 1989, and, in some cases, 

reflect a precautionary approach that was based on the more limited scientific understanding of 

the disease at the time. As new scientific findings 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 regulations 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 again 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 boneless 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 continue 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 classification structure (i.e., negligible, controlled, or undetermined 

risk) to more accurately represent 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 States participates in the 

process of reviewing the BSE Code and is supportive of those changes that provide a sciencebased framework facilitating international trade. 

This supporting scientific document summarizes the significant components of the relevant BSE 

Code chapter and the scientific information that supports the revised Code chapter. The key 

components include the three levels of risk status classification for countries, the risk evaluation 
3 

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 addition 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 (PrPSc) is both less soluble and more resistant to 

degradation than the normal form (Taylor 2000; Taylor et al. 1995). The PrPSc is extremely 

resistant to heat and to normal sterilization processes, making it difficult to inactivate with 

standard methods used to process human food and animal feed. Although rendering and other 

processes can partially inactivate PrPSc, the risk mitigation strategies (for meat and meat 

products) rely mainly on the elimination 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 food chain eliminates more than 99 percent of the 

potential infectivity from infected carcasses, thereby preventing consumer exposure through 

foods. Prohibiting their consumption provides a substantial reduction in risk to consumers in 

countries where BSE has been shown to exist and 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 appendix (Wells et al. 1994, 

1996, 1998, and 1999). 

Approximately 90 percent of the infectivity is associated with the brain, spinal column, dorsal 

root ganglia, and trigeminal ganglia. The remaining 10 percent is associated with the distal 

ileum. In BSE as in other TSEs, the total amount of infectivity in an animal increases throughout 

the incubation period, reaching the highest load very close to the death of the animal. Infectivity 

is considered to increase exponentially, reaching 3 logs less than a clinical case 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). Consequently, what becomes an SRM is dependent on the age of the 

animal. The next paragraphs briefly describe infectivity 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 expected 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 Bradley 2003; Hoffman et al. 2007). 

 Spinal cord – Studies in experimentally and naturally infected cattle demonstrate that the 

spinal cord of BSE-infected cattle is expected to be infectious in the later stages of the 

incubation period (Wells et al. 1998, 1999, and 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 studies where cattle were inoculated intra 

cerebrally (cattle bioassay) with 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 other sections of the 

intestine. In addition, in naturally occurring 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 pathogenesis study (Hoffman et al, 2011) found 

high amounts of infectivity in the distal ileum, 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 orally with titred BSE brainstem (Okada, 2011), PrPsc was 

confirmed in the distal ileum and the distal jejunum by immunohistochemistry 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 animals exposed to high doses of the agent could 

extend to the jejunum and the duodenum Given that the cattle in the two pathogenesis 

studies described above were exposed to a very high dose of infectivity that represents 

levels that exceed natural field exposure conditions, intestine other than the distal ileum is 

highly unlikely to contain epidemiologically significant 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 derived jejunum could contain any infectivity is 

insignificant. Therefore the likelihood that any cattle could be exposed to any infectivity 

from the jejunum is extremely low; so low as to be considered negligible. Additionally, 

the international community has not changed its 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 incubation 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. 1998 and 2005; Terry et al. 2003). 

 Skull – Because of the skull’s association with brain and eyes, with resultant 

contamination through the slaughtering process or because of residual brain tissue 

following removal of the brain, the skull is considered to be SRM in the later stages of 

the incubation period. 

 Vertebral column – Because of a combination of close association with dorsal root 

ganglia and the superficial contamination of the cut surface of the spine with spinal cord 

during the carcass splitting process, the vertebral column is considered to be SRM in the 

later stages of the incubation period. 
5 

The agent does not evoke a traditional immune response or inflammatory reaction 

(Khalili-Shirazi et al. 2005); thus, reliable ante-mortem diagnostic tests based on host reaction 

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

tissue or detection of PrPSc 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; Casalone et al. 2004; DeBosschere 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 several 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” BSE 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 World Trade Organization as the 

standard-setting body charged with developing science-based recommendations for trade in 

animals and animal products. The OIE publishes its standards in the Terrestrial Animal Health 

Code. Unless there is scientific justification for not doing so, Member Countries 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 risk 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 among countries but also establishes risk mitigation 

measures for trading in live animals and products, even from countries 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 dossier 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 could 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 effective ruminant-to-ruminant feed ban). A 

requesting country must also submit information on its awareness program and notification 

requirements for BSE, its diagnostic capabilities, and its BSE surveillance program. The 

information in this dossier is evaluated by leading BSE experts from OIE member countries to 
6 

determine the country’s BSE risk status classification, which in turn will determine import 

requirements for live cattle and cattle products that are not designated for unrestricted trade. 

To retain OIE’s classification of disease status, a country must continue to observe OIE’s 

guidelines and report any significant events that might change that status (OIE 2006). OIE 

reserves the right to revoke 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 risk unless the criteria of Article 11.6.3 continue 

to be met. For reinstatement of negligible risk status, the country should submit a dossier of 

explanatory data to the OIE as quickly as possible and at the latest 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 demonstrated that appropriate measures have been 

taken for a specific time and have managed the risks. Live cattle and cattle commodities 

originating from negligible-risk countries are considered 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 demonstrated that appropriate specific measures have been taken 

for the relevant period of time defined below to manage each identified risk; 

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

3. EITHER: 

a. There has been no case of BSE identified within the country, or, if there has been a 

case, every case of BSE has been demonstrated to have been imported and has been 

completely destroyed, and 

i. Appropriate awareness and notification programs and laboratory diagnostic 

procedures have been in place for at least 7 years;2

 and 

ii. It has been demonstrated through an appropriate level of control and audit that 

for at least 8 years3

 neither meat-and-bone meal nor greaves derived from 

ruminants has been fed to ruminants; 

 

2

 The 7-year requirement reflects the 95th 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 notification programs and laboratory diagnostic 

procedures have been in place for at least 7 years; and 

ii. It has been demonstrated through an appropriate 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 appropriate 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 are necessary for trade in live cattle or cattle 

products from a country designated as controlled risk. 

The following criteria must be met for a country 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 that 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 adequate surveillance has been carried out; and 

3. EITHER: 

a. There has been no case of BSE or, if there has been a case, every case of BSE has 

been demonstrated to have been imported and has been completely destroyed, 

appropriate awareness and notification programs and laboratory diagnostic 

procedures are in place, and it can be demonstrated through an appropriate level of 

control and audit that neither meat-and-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 programs and laboratory diagnostic 

procedures have not been in place for at least 7 years; 
8 

ii. It cannot be demonstrated that controls over the feeding 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 that neither meat-and-bone meal nor greaves 

derived from ruminants has been fed to ruminants, but at least one of the following 

two conditions applies: 

i. Awareness and notification programs and laboratory diagnostic procedures 

have not been in place for at least 7 years; 

ii. It cannot be demonstrated that controls over the feeding 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 undetermined BSE risk if it cannot be demonstrated 

that it meets the requirements of another category. Either the country has undergone the 

evaluation process and has not met the conditions for 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 classification 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 BSE occurrence in the country. A release assessment and, if justified, 

an exposure assessment may be part of that risk assessment. 

The release assessment should address the likelihood that the BSE agent already exists or has 

been introduced into the exporting country through the importation of potentially infective cattle 

or contaminated cattle products. 
9 

The exposure assessment should assess the likelihood of exposure of the BSE agent to cattle in 

the exporting country. The exposure assessment should include information on the following: 

 the likelihood of recycling and amplification of the BSE agent through consumption by 

cattle of meat-and-bone meal or greaves of ruminant origin or other feed or feed 

ingredients contaminated with these; 

 the origin and use of cattle carcasses (including fallen stock), byproducts, and 

slaughterhouse waste; 

 the parameters of the rendering processes, cattle feeding practices, and the methods of 

cattle/animal feed production 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 history of BSE in the exporting country. 

Release assessment of the exporting country 

The exporting country’s release assessment 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 exporting 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 present a higher release risk than those originating 

from low-risk countries. Exporting countries must 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 country and in what quantities. 

Potentially infected live cattle 

Assessing the risk of the release of the BSE agent in the exporting country through the 

importation of potentially infected live cattle into the country relies on several underlying 

assumptions (OIE 2009). The most significant of these is that the risk of BSE transmission is 

influenced by the date at which imports occurred (relative to the BSE mitigations in the country 

of origin) and is proportional to the volume of potentially contaminated imports. 
10 

Based on these assumptions, the risk of the release 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 the 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 within the context of these risk factors about the 

importation of live cattle during the previous 7 years. 

Products of animal origin potentially infected with BSE 

To assess the risk of the release of the BSE agent from an exporting 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 agent in the exporting country is 

dependent on: 

 The species of origin of the animal products and whether these products 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 animals 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 livestock even if meat-and-bone 

meal and greaves, or feedstuffs containing 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 products of animal origin during the previous 7 years. 

Exposure assessment of the exporting country 

The exposure assessment assesses the likelihood of exposure of the BSE agent to cattle in the 

exporting country. The exposure assessment should include information on the potential for the 
11 

exposure of cattle to the BSE agent through consumption 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 feed; 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/animal feed production. 

The potential for the exposure of cattle to the BSE agent through consumption of meat-and-bone 

meal or greaves of bovine origin 

According to scientific literature, cattle consumption of meat-and-bone meal or greaves of 

ruminant origin plays the main role in BSE transmission. 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 unlikely. (Countries applying 

for negligible risk status are required to demonstrate that the 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 carcasses, by-products, and slaughterhouse waste; the parameters of the 

rendering processes; and the methods of cattle feed production 

Evidence indicates that the transmission of BSE requires 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 animal, carrying significant amounts of the infectious 

agent, must die or be slaughtered; tissues from that animal that contain the infectious agent must 

be sent to a rendering facility; the infectivity present 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 the risk of transmitting BSE infectivity, not only 

because of its role in inactivation of the BSE agent but also because it serves as a critical control 

point for the redirection of ruminant protein away from cattle feeds. If a fraction of the BSE 

infectivity were to escape destruction at the rendering facility, it would need to bypass controls 

imposed to prevent cross-contamination 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 the cattle population of a country is proportional to the level of known 

or potential exposure to BSE 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 cattle 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 byproducts, 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 history of the country, 

including surveillance data. Surveillance 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. Surveillance 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 country, the goal of BSE surveillance could be to: 

 Detect cases at a predetermined design prevalence; 

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

 Monitor the effectiveness of a feed ban and/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 collecting as many samples as 

possible from the surveillance stream that has 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 with the next highest likelihood for finding BSE: 

animals that are nonambulatory or condemned ante-mortem at slaughter (casualty or emergency 

slaughter), followed by cattle which are found dead (fallen 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 sample those cattle populations where 

the disease is most likely to be found (USDA 2006). 

OIE has identified the following four subpopulations of cattle for surveillance purposes: 
13 

1. Clinical suspects: Cattle over 30 months old displaying behavioral or clinical signs 

consistent with BSE. 

2. Casualty slaughter: Cattle over 30 months, nonambulatory, recumbent, unable to rise or 

to walk without assistance, sent for emergency slaughter or condemned at ante-mortem 

inspection. 

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

an abattoir. 

4. Healthy slaughter: Cattle over 36 months exhibiting 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 country with an adult cattle 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 recommended 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 conclusions 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 also conduct Type B surveillance after meeting the points target 

using Type A surveillance to maintain confidence 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 samples 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, the curriculum, how long the program has been in place, and any 

contingency and/or preparedness plans that address BSE. 

Compulsory notification and investigation 

The socioeconomic implications associated with BSE require that there be incentives and/or 

obligations to report and investigate suspect cases. Therefore, countries must provide 

information about guidance given to veterinarians, producers, workers at auctions, 

slaughterhouses, etc. with regard to the criteria that would initiate the investigation of an animal 

as a BSE suspect; whether these criteria have changed 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 payments or penalties for not reporting a suspect. 
14 

Sample testing 

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

accordance with the OIE’s Manual of Diagnostic Tests and Vaccines 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 classification of disease status, a 

country must continue to observe OIE’s guidelines and report any significant events that might 

change that status. The OIE reserves the right to revoke the given 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 epidemiological 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 and cattle products on two criteria: the risk 

classification status of the exporting country and the risk of BSE transmission posed by the 

various products. This section discusses the risk associated with specific commodities with 

regard to BSE transmission, including experimental data, epidemiological 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 the risk associated with trade of the specific commodity as well 

as any conditions necessary for trade based on the risk classification 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 regardless 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 insoluble 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; and 
15 

 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 for 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 certification requirements are in place through separate import 

protocols. Therefore, the scientific literature relevant to this commodity is discussed here. 

Commodities for restricted trade 

Other commodities that have been shown to pose 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 unprocessed material for the preparation of cosmetics, drugs 

(including pharmaceuticals and natural health products) or medical devices; 

 Tallow and tallow derivatives (other than tallow 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, pharmaceuticals including biologicals, or medical 

devices; and 

 Meat-and-bone meal. 

Specified risk materials (SRM) 

Conditions for trade in several of the restricted commodities include removal of SRM. These 

materials pose the greatest risk of containing the 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 vertebral column from animals over 12 months of age from undeterminedrisk countries. Although the skull and vertebrae themselves do not carry infectivity, they are 

considered risk materials because of their association with infective tissues. 

Similarly, in the United States, the Food Safety and 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 use 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 vertebral column, the vertebrae of the tail, 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 the 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 countries 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 the infectious dose received, i.e., the larger an 

infectious dose received, the shorter the incubation 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 infectivity. 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 1989, was 21 months. The detection 

of BSE in young animals during the peak of the BSE epidemic in the UK supports a cautious 

approach in defining SRMs for regions of undetermined 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 management measures. Because BSE infectivity is 

detectable in central nervous 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 since the start of BSE surveillance 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 (Middleton and Barlow 1993; Barlow and Middleton 

1990; Bradley 1990), nor in placenta, placental fluids, or ovary or uterine caruncle following 

mouse inoculation (Fraser and Foster 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 placenta 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 with clinical BSE) was used for artificial 

insemination of 167 clinically affected cows in the terminal stages of BSE. The resulting 

embryos were treated according to the recommendation of the International Embryo Transfer 

Society (IETS). The embryos were always transferred singly, but repeat transfers were done if 

returns to estrus occurred within the particular transfer session. Researchers transferred 587 

viable embryos into 347 recipient heifers imported 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. Twenty-seven heifers and 20 

offspring died during monitoring but none showed signs 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 scrapie-associated fibrils. All 
17 

results were negative. In addition to the embryos transferred into recipient heifers, 1,020 

nonviable embryos were inoculated intracerebrally 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 intracerebral and intraperitoneal inoculation of 946 

mice. One of these mice had some vacuolar pathology, but its relevance proved difficult to 

determine because the putative incubation period was 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. Results of the study indicate that embryos are 

unlikely to carry BSE and do not transmit the disease to recipients and their embryo transfer 

offspring, even when they are collected from donor cows at the end-stage when the risk of 

maternal transmission (if it were to exist) could potentially 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 without BSE from the same farm and age cohort 

(controls) have been observed under controlled conditions over a 7-year period. The purpose of 

the study was to determine whether maternal transmission occurs, and, if so, at what level of 

incidence. There was a statistically significant risk difference between 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 relative 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 disease 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 susceptibility to feed exposure that could have 

occurred in any cattle in the study) and genuine transmission best fits the computer model of the 

epidemic (Donnelly et al. 1997). Later studies by Donnelly et al. (2002) significantly reduced the 

estimated risk to offspring, although the authors recognized that the introduction 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 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. 

OIE guidelines. Experimental and epidemiological evidence 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 collected and handled in accordance with the 

recommendations of the IETS should be unrestricted, regardless of the BSE status of the 

exporting region. 

Gelatin 
18 

Supporting documentation. Gelatin is a highly purified protein manufactured 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 steps of the refining process, as well as the 

resulting infectivity reduction, are 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 present 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 collagen protein and is believed to decrease the 

infectivity titer that might be present (Grobben et al. 2004). 

3. Alkaline treatment: The materials are soaked in a saturated lime solution (pH 12.5) for 20 

to 50 days. The alkaline treatment changes the internal structure of the BSE protein, if 

present. The combination of time, temperature, and concentration of the alkaline 

treatment reduces the titres of BSE infectivity if present in the raw 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 performed, gelatin is extracted by a series 

of hot water steps. These include purification by filtration and sterilization, both of which 

further remove suspended materials and thus further reduce the level of any remaining 

BSE infectivity, if present, which is unlikely at this stage in the production. 

Research studies mimicking the manufacturing process described above were unable to show 

detectable levels of infectivity in the mouse bioassay. The results are consistent with TSE 

infectivity reduction capacity, exceeding a factor of 30,000 (4.5 logs, although results from most 

recent research indicate clearance factors exceeding 4.8 logs) (EC SSC 2003a). These studies 

have demonstrated that the common process of manufacturing bovine gelatin provides 

significant assurance of low risk for BSE. 

A quantitative risk assessment by the European 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 different risk levels. The study did not take into consideration the 

sourcing of bones. Results indicate that the relevant exposures are 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 vertebral 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 the healthy slaughter subpopulation 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-derived gelatin that is produced 

using common manufacturing processes could be considered safe regardless 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 gelatin and collagen derived from bones can be 

traded based on manufacturing practices and the following conditions: 

1. Negligible-risk countries: Gelatin and collagen derived from bones can be traded freely 

provided that cattle pass ante-mortem and post-mortem inspection. 

2. Controlled and undetermined-risk countries: Gelatin and collagen derived from bones can 

be traded provided that (1) cattle passed ante-mortem and post-mortem inspection; (2) the 

vertebral column from cattle over 30 months of age at the time of slaughter and skulls are 

excluded from processing; and (3) the bones are subjected to the process listed in OIE 

Code Article 11.6.15 or equivalent.

Tallow 

 

Supporting documentation. Several studies have evaluated TSE infectivity in tallow that was 

spiked with infected brain material and then subjected to rendering. In two rendering studies, one 

with BSE (Taylor et al. 1995) and the other with scrapie (Taylor et al. 1997), no detectable 

infectivity to either agent was demonstrated from any of the tallow samples when assayed in 

mice. The BSE rendering study did not demonstrate any infectivity in crude unfiltered tallow, 

although the same rendering procedure produced meat-and-bone meal with almost the same 

infectivity titer levels as the untreated raw material. These studies suggest that during the 

manufacturing process of tallow, both BSE and scrapie 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 agents 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 studies confirmed the lack of detectable infectivity of tallow 

through mice bioassay, and (2) epidemiological studies were not able to link 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 epidemiological report on BSE in Japan hypothesized 

that tallow in calf milk replacer was one possible source and route of infection. However, 

statistical analysis of the data did not support any correlation between the use of milk replacer 

and BSE incidence (Ministry of Agriculture, Forestry, and Fisheries 2003). A more recent 

retrospective study of BSE cases in Japan implicated milk replacers, 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 cluster, thus leading the authors 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 effect 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 high level of uncertainty, the study found that under 

certain scenarios tallow-based milk replacer could be associated with transmission of BSE to 
20 

calves. The simulations demonstrated the importance of SRM exclusion in limiting the 

probability of BSE infection, particularly from tallow with high impurity levels (0.5 percent). 

Uncertainty in the results stemmed from infectivity 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 risk of exposure, they concluded that the source 

of raw material warranted further consideration. In addition, removal 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 with a maximum level of insoluble impurities of 0.15 

percent by weight and derivatives made from this tallow can be unrestricted regardless of BSE 

risk status of the cattle population. 

Tallow with more than 0.15 percent insoluble impurities by weight requires certification that: 

 It is sourced from a negligible-risk country; or 

 If it is sourced from a controlled-risk country, it is derived from cattle that have passed 

ante-mortem and post-mortem inspections and does not contain SRM. 

Tallow derivatives 

Supporting documentation. Several studies have evaluated the effects of various time, 

temperature, and pressure processes on prion inactivation. As discussed in the previous section, 

the BSE-spiked tallow study (Taylor et al. 1995) showed that while infectivity does not persist in 

tallow during rendering, the level of infectivity of BSE subjected to a poorly inactivated 

rendering process remained at almost the same level as the untreated material. 

Tallow derivatives are generally produced using manufacturing processes that include 

hydrolysis, saponification, or transesterification under high temperature and pressure conditions. 

Products made from this process have been shown 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 transit of tallow derivatives other than those made from tallow 

with a maximum level of insoluble 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 inspections and does not contain SRM; or 

 The tallow is produced by hydrolysis, saponification, or transesterification under high 

temperature and pressure conditions. 

Dicalcium phosphate 
21 

Supporting documentation. Considerable mineral content is recovered from the hydrochloric acid 

treatment of bone chip used in the production of gelatin. As mentioned earlier, before bones can 

be used in the manufacture of gelatin, fat and 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 soft tissues. Before degreased bone chip material 

can be utilized to produce gelatin, minerals (including calcium and phosphate) must be removed. 

To remove minerals, the bone chip is soaked in hydrochloric acid (approximately 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 resultant 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 pretreatment of bone during 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 manufacturing process. Further, a 

significant reduction of TSE infectivity under experimental 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 validation study by Grobben et al. shows that the acid 

process after degreasing and demineralization as described in the gelatin manufacturing process 

has a total clearance of infectivity of 2.6 log10 (EC SSC 2003). The production process as a 

whole reduces the infectivity further up to 3.8 to 3.9 log10, resulting 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 bones with dorsal root ganglia and spinal cord, a 

very low infectivity of bone marrow, and an infectivity reduction for the acid process of 104.2 – 

104.8 and for the heat/pressure process of 106.2 – 106.8 (OIE Terrestrial Animal Health Standards 

Commission [TAHSC] 2006). 

In 2003, the EC SSC stated that the residual risk 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 BSE 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-contamination 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 protein or fat. Dicalcium phosphate produced under 

such conditions can be traded without 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 controlled BSE risk, it must be derived from 

cattle that have passed ante-mortem and post-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 and greaves are considered to be one of the 

highest risk commodities for BSE. OIE recommends that ruminant meat-and-bone meal and 

greaves (or any products containing these commodities) can be traded only under the following 

conditions: 

 Products originate from a country classified as negligible risk; and 

 Bovines from which the product originated were born after the date the ban was 

effectively enforced, if the exporting country 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 infectivity has not been demonstrated in the muscle tissue of 

BSE-infected cattle examined in either the mouse bioassay or in cattle 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 small ruminants infected with TSEs other than BSE 

(Bosque 2002). Those findings are consistent with differences in the transmission, host range, 

genetic susceptibility, infectivity distribution, and epidemiology found in different TSEs that 

affect animals and humans. However, in transgenic mice that overexpress the bovine PrP gene 

(Tg bov XV), infectivity was detected in one muscle (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 reported 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 experimental BSE in cattle, no infectivity is found in 

assays of skeletal muscle pools (triceps, masseter, sternocephalicus, and longissimus dorsi) 

completed in wild-type mice bioassay and in cattle bioassay of skeletal muscle pools (masseter, 

semitendin-osus, and longissimus dorsi) from selected 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 cattle challenged orally with BSE; culled at 20, 

24, 27, 30, and 33 months; and inoculated intracerebrally into BoPrP-Tg110 mice have failed to 

detect infectivity in muscle (Espinosa 2007). 

The international scientific community largely considers 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 infectivity in muscle for other TSEs 

in different species in relation to human food safety. EC SSC concluded that there was no reason 

to revise its opinions regarding the safety of meat, given the consistent negative results in BSE 

infectivity experiments (EC SSC 2002d). SEAC concluded 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 recommended stunning practices (EFSA 2004; International 

Forum for Transmissible Animal Diseases and Food Safety 2009) and the hygienic removal of 

SRM (EFSA 2005), skeletal muscle meat itself is considered safe. 

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

blood does not constitute a risk of BSE transmission (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 contamination of the beef with the BSE agent is 

unlikely to occur. These controls are directed toward procedures 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 force visible pieces of brain and other CNS tissue 

into the circulatory system of stunned cattle. In addition, the pithing process could cause 

dissemination of central nervous tissue throughout the body. Avoiding these procedures 

eliminates the potential of this risk. Finally, SRM must be handled in ways that prevent 

contamination of the carcass since, their removal 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, OIE 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 process 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 elongated 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 products, which are the muscle and parts that can 

include but are not limited to certain organs of a slaughtered animal 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 indigenous cases of BSE, the cattle from which 

the fresh meat and meat products were derived were born after the date the ban on the 

feeding of ruminants with meat-and-bone meal and greaves derived from ruminants 

has been effectively enforced. 

For countries classified as controlled BSE risk: 

a. Cattle were not subjected to a stunning process 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 contain 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 countries (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 process 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 contain 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 with nervous and lymphatic tissues exposed 

during the deboning process. 

Blood and blood products 

Blood and blood products can be divided into two main groups: whole blood and cellular 

derivatives such as red cell concentrate, platelets 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 the blood. Serum is plasma with fibrinogen and 

clotting factors removed. 
25 

Supporting documentation. BSE infectivity was not demonstrated 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 intraperitoneally 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, fetal 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. However, 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) utilizing 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 from clinically normal infected 

sheep, caused disease in 2 of 24 recipients. Blood or buffy coat taken 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 sheep out of 21 transfused with blood 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 examined these studies and their implications and 

concluded that the finding of infectivity in the blood of sheep could not be extrapolated to BSE 

in cattle (EC SSC 2002c). 

Brown et al. (1999), using a human strain of TSE in mice inoculated intracerebrally, concluded 

that infectivity was present in the buffy coat during 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 plasma was found, but still 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 scrapie or CJD demonstrated that the fractions 

containing white blood cells have the highest levels of infectivity. 

In contrast to investigations of the natural distribution of infectivity in rodent blood fractions, 

one “spiking” study added high levels of hamster-adapted scrapie infectivity from brain 

homogenate to normal human blood. Following fractionation by centrifugation into red cells, 

white cells/platelets, and plasma components, titrations 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 distribution of TSEs in blood, may potentially apply to the distribution 

following cross-contamination at blood collection. Therefore, if any BSE infectivity is present in 

bovine blood, either naturally or via cross-contamination, it would likely be highest in the 

cellular components. These fractions, both red and white cells, are excluded when harvesting 

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

Further decrease in TSE infectivity occurs with fractionation of plasma proteins. Fractionation is 

the process whereby specific proteins, such as albumin, are separated from other components of 
26 

the plasma. Infectivity in various fractions has been examined. For example, using data from 

several cited studies, Comer (2004) estimated that human albumin contains 3.1  10-5 vCJD 

ID50/gram. Compared to Comer’s estimates of infectivity in whole blood (2 iv vCJD ID50/gram), 

this figure represents a dramatic decrease. 

The possibility of cross-contamination of blood products with SRM exists at the time of 

collection, particularly in a slaughter environment. Certain slaughterhouse stunning practices 

specifically the use of devices that inject compressed air or gas 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, collection of blood in an open manner may allow other 

tissues to contaminate the blood. 

OIE guidelines. To prevent contamination due to such potential sources 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 slaughter, 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 only 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 demonstrated the effectiveness of implementing a 

ruminant-to-ruminant feed ban on significantly reducing the prevalence of BSE. 

The 165 cases of BSE born in the UK after the implementation 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 2001. 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 negligible and controlled BSE risk countries should 

be allowed under the following conditions: 

a. For negligible BSE risk: Animals are identified 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 case 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 complies with the conditions under Article 11.6.4, 

animals are identified by a permanent identification system in such a way that 
27 

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 countries can export live cattle if the country has 

an effectively enforced ruminant-to-ruminant feed ban and the cattle selected for export were 

born 2 years after the date the ban was effectively enforced. Trade of live bovines from 

undetermined-risk countries should 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 was 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 these 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 ban — such as veterinary infrastructure, surveillance, and import 

controls — play a role. Such factors can be taken into consideration if a country has been 

categorized for BSE risk. When an undetermined-risk country demonstrates that an effectively 

enforced feed ban is in place and confirms that the other conditions have been met (both 

necessary for exporting live bovines), APHIS believes 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 bovines 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, necessitating 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 importation 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 exhibitions, 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 exporting country to show that they have not been 

exposed to any BSE cases (i.e., are not exposed cattle). Exposed cattle 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 international veterinary certificate must describe 

the permanent identification system that demonstrates that the cattle to be exported are not 

exposed cattle. 
29 

References

Anil, M. H., S. Love, S. Williams, A. Shand, J. L. McKinstry, C. R. Helps, A. WatermanPearson, J. Seghatchian, and D. A. Harbour. 1999. Potential contamination of beef 

carcasses with brain tissue at slaughter. Vet. Rec. 145: 460-462. 

APHIS. Revised assessment of bovine spongiform encephalopathy (BSE) risks associated with the 

importation of certain commodities from BSE minimal risk regions (Canada). 2007. 

(www.aphis.usda.gov/newsroom/hot_issues/bse/downloads/risk_assessment_%20final9-2007.pdf) 

Appel, T., M. Wolff, F. von Rheinbaben, M. Heinzel, and D. Riesner. 2001. Heat stability of prion rods 

and recombinant prion protein in water, lipid and lipid–water mixtures. J. Gen. Virol. 82: 465-473.

Balkema-Buschmann A, Eiden M, Hoffmann C, Kaatz M, Ziegler U, Keller M, Groschup MH. BSE 

infectivity in the absence of detectable PrP(Sc) accumulation in the tongue and nasal mucosa of 

terminally diseased cattle. J Gen Virol. 2011 Feb;92(Pt 2):467-76 

Barlow, R.M. and D.J. Middleton. 1990. Dietary transmission of bovine spongiform encephalopathy to 

mice. Vet. Rec. 126: 111-112. 

Béringue, V., A. Bencsik, A. Le Dur, F. Reine, T. Lan Laï, N. Chenais, G. Tilly, A-G. Biacabé, T. Baron, 

J.L. Vilotte, and H. Laude. 2006. Isolation from Cattle of a Prion Strain Distinct from That Causing 

Bovine Spongiform Encephalopathy, PLoS Pathog. 2(10): e112. 

Bolton D.C., M.P. McKinley, and S.D. Prusiner. 1982) Identification of a protein that 

purifies with the scrapie prion. Science. 218(4579): 1309-11. 

Bosque, P.J., C. Ryou, G. Telling, D. Peretz, G. Legname, S.J. DeArmond, and S.B. Prusiner. 

2002. Prions in skeletal muscle. Proc. Natl. Acad. Sci. 99: 3812-3817. 

Bradley, R. 1990. Editorial: Bovine Spongiform Encephalopathy: The Need for Knowledge, Balance, 

Patience, and Action. J. Pathol. 160: 283-285. 

Bradley, R. 1996. The research programme on transmissible spongiform encephalopathies in Britain with 

special reference to bovine spongiform encephalopathy. Dev. Biol. Scand. 80: 

157-170. 

Brown, P., R.G. Rohwer, B.C. Dunstan, C. MacAuley, D.C. Gajdusek, and W.N. Drohan. 1998. The 

distribution of infectivity in blood components and plasma derivatives in experimental models of 

transmissible spongiform encephalopathy. Transfusion 38: 810-816. 

Brown, P., L. Cervenakova, L.M. McShane, P. Barber, R. Rubenstein, and W.N. Drohan. 

1999. Further studies of blood infectivity in an experimental model of transmissible spongiform 

encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt-Jakob disease 

in humans. Transfusion 39: 1169-1178. 

Buschmann A., A. Gretzschel, A.G. Biacabe, K. Schiebel, C. Corona, C. Hoffmann, M. Eiden, T. Baron, 

C. Casalone, M.H. Groschup. 2006. Atypical BSE in Germany — proof 

of transmissibility and biochemical characterization. Vet. Microbiol. 117(2-4): 103-116. Epub August 17. 
30 

Buschmann, A., A. Gretzschel, A.G. Biacabe, K. Schiebel, C. Corona, C. Hoffmann, M. Eiden, T. Baron, 

M. Casalone, F. Tagliavini, J. Langeveld, and M.H. Groschup. 2006a. Characterization of atypical BSE 

cases of the hand l-type in Germany. Poster at Prion2006, Turin, October 4-6, 2006. 

Buschmann, A., and M. Groschup. 2005. Highly bovine spongiform encephalopathy-sensitive transgenic 

mice confirm the essential restriction of infectivity to the nervous system in clinically diseased cattle. J. 

Infect. Dis. 192: 934-942. 

Casalone, C., G. Zanusso, P. Acutis, S. Ferrari, L. Capucci, F. Tagliavini, S. Monaco, and M. Caramelli. 

2004. Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular similarities 

with sporadic Creutzfeldt–Jakob disease. Proc Natl Acad Sci 101(9): 3065-3070. 

Comer, P.J. and P.J. Huntly. 2003. Exposure of the human population to BSE infectivity over the course 

of the BSE epidemic in Great Britain and the impact of changes to the over thirty month rule. Over Thirty 

Month Rule review paper. Accessed September 7, 2007. 

(www.food.gov.uk/multimedia/pdfs/otmcomer.pdf) 

Comer, P.J. 2004. Risk assessment of exposure to vCJD infectivity in blood and blood products. 

Department of Health, U.K. (www.dnv.com/binaries/vCJD_Update_Report_tcm4-74414.pdf) 

De Bosschere, H., S. Roels, E. Vanopdenbosch, A. Buschmann, A. Gretzschel, A.G. Biacabe, K. 

Schiebel, C. Corona, C. Hoffmann, M. Eiden, T. Baron, C. Casalone, and M.H. Groschup. 2004. Atypical 

case of bovine spongiform encephalopathy in an East-Flemish cow in Belgium. Intern. J. Appl. Res. Vet. 

Med. 2(1): 52-54. 

Donnelly, C.A., S. Gore, R. Curnow, J.W. Wilesmith. 1997. The bovine spongiform encephalopathy 

maternal cohort study: Its purpose and findings. Appl. Statistics 46(3): 299-304. 

Donnelly, C.A., N.M. Ferguson, A.C. Ghani and R.M. Anderson. 2002. Implications of BSE infection 

screening data for the scale of the British BSE epidemic and current European infection levels. Proc. Biol. 

Sci. 269(1506): 2179-2190. 

Espinosa, J.C., M. Morales, J. Castilla, M. Rogers, and J.M. Torres. 2007. Progression of prion infectivity 

in asymptomatic cattle after oral bovine spongiform encephalopathy challenge. J. Gen. Virol. 88: 1379-

1383. 

EC SSC. 2000. Opinion on the safety of ruminant blood with respect to TSE risks. Adopted at its meeting 

of 13-14 April 2000. (http://ec.europa.eu/food/fs/sc/ssc/out74_en.pdf)

EC SSC. 2002a. Update of the opinion on TSE infectivity distribution in ruminant tissues. 

(http://ec.europa.eu/food/fs/sc/ssc/out296_en.pdf) 

EC SSC. 2002b. The safety of bovine embryos. Amendment to the SSC opinion of 18-19 March 1999 on 

the possible vertical transmission of bovine spongiform encephalopathy (BSE). Adopted by the Scientific 

Steering Committee at its meeting of 16 May 2002. (http://ec.europa.eu/food/fs/sc/ssc/out263_en.pdf) 

EC SSC. 2002c. The implications of the recent papers on transmission of BSE by blood transfusion in 

sheep (Houston et al. 2000; Hunter et al. 2002) adopted by the Scientific Steering Committee at its 

meeting of 12-13 September 2002. (http://ec.europa.eu/food/fs/sc/ssc/out280_en.pdf) 
31 

EC SSC. 2002d. Prions in muscle statement adopted by the scientific steering committee at its meeting of 

4-5 April 2002. (http://ec.europa.eu/food/fs/sc/ssc/out254_en.pdf) 

EC SSC. 2003a. Updated opinion on the safety with regard to TSE risks of gelatine derived from 

ruminant bones or hides. Adopted by the Scientific Steering Committee at its meeting of 6-7 March 2003. 

(http://ec.europa.eu/food/fs/sc/ssc/out321_en.pdf) 

EC SSC. 2003b. Updated opinion and report on the safety of dicalcium phosphate (DCP) And tricalcium 

phosphate (TCP) from bovine bones used as an animal feed additive or as fertiliser. Submitted to the 

Scientific Steering Committee at its meeting of 6-7 March 2003. Accessed December 20, 2007. 

(http://ec.europa.eu/food/fs/sc/ssc/out322_en.pdf) 

EFSA. 2004. Opinion of the EFSA on BSE risk from dissemination of brain particles in blood and carcass 

following stunning. EFSA Journal 123: 1- 4. 

EFSA. 2005. Opinion on the assessment of the human and animal BSE risk posed by tallow with respect 

to residual BSE risk. EFSA Journal 221: 1-47. 

EFSA. 2006. Quantitative assessment of the human and animal BSE risk posed by gelatine with respect to 

residual BSE risk. Adopted on 18 January 2006. EFSA Journal 312: 1-29. 

Fraser, H. And J.D. Foster. 1994. Transmission to mice, sheep and goats and bioassay of bovine tissues. 

Transmissible Spongiform Encephalopathies. Proceedings of a Consultation with the Scientific 

Veterinary Committee of the CEC. R. Bradley and B. Marchant, eds. VI/413/94-EN Brussels, EC. pp 

145-149. 

FSIS. 2005. Federal Register Volume 70, No. 173, pp. 53043-53050. Interim final rule: Prohibition of the 

use of specified risk materials for human food and requirements for the disposition of non-ambulatory 

disabled cattle.” Docket 03-025IFA. September 7. 

Grobben A., P. Steele, R. Somerville, and D.M. Taylor. 2004. Inactivation of the bovine spongiform 

encephalopathy (BSE) agent by the acid and alkaline processes used in the manufacture of bone gelatine. 

Biotechnol. Appl. Biochem. 39: 329-338. 

Grobben A., P. Steele, R. Somerville, and D.M. Taylor. 2006. Inactivation of transmissible spongiform 

encephalopathy agents during the manufacture of dicalcium phosphate from bone. Vet. Rec. 158: 361-

366. 

Hill, W. 2005. Review of the Evidence for the Occurrence of ‘BARB’ BSE Cases in Cattle. Accessed 

September 7, 2007. (www.defra.gov.uk/animalh/bse/pdf/hillreport.pdf) 

Hoffmann, C., U. Ziegler, A. Buschmann, A. Weber, L. Kupfer, A. Oelschlegel, B. Hammerschmidt, and 

M. Groschup. 2007. Prions spread via the autonomic nervous system from the gut to the central nervous 

system in cattle incubating bovine spongiform encephalopathy. J. Gen. Virol. 88: 1048-1055. 

Hoffmann C, Eiden M, Kaatz M, Keller M, Ziegler U, Rogers R, Hills B, Balkema-Buschmann A, van 

Keulen L, Jacobs JG, Groschup MH. BSE infectivity in jejunum, ileum and ileocaecal junction of 

incubating cattle. Vet Res. 2011 Feb 7;42(1):21 
32 

Houston, F., J. Foster, A. Chong, N. Hunter, and C. Bostock. 2000. Transmission of BSE by blood 

transfusion in sheep. Research letter. The Lancet. 356: 999-1000. 

Hunter, N., J. Foster, A. Chong, S. McCutcheon, D. Parnham, S. Eaton, C. MacKenzie, and F. Houston. 

2002. Transmission of prion diseases by blood transfusion. J. Gen. Vir. 83: 2897-2905. 

International Forum for Transmissible Animal Diseases and Food Safety. 2009. Position paper on 

slaughter practices and the dangers of carcass contamination with BSE. 

(www.tseandfoodsafety.org/position_papers/TAFS_POSITION_PAPER_ON 

%20SLAUGHTER%202009.pdf) 

Khalili-Shirazi, A. S. Quaratino, M. Londei, L. Summers, M. Tayebi, A.R. Clarke, S.H. 

Hawks, G.S. Jackson, and J. Collinge. 2005. Protein conformation significantly influences immune 

responses to prion protein. J. Immunol. 174(b): 3256-63. 

Lloyd, S.E., J.M. Linehan, M. Desbruslais, S. Joiner, J. Buckell, S. Brandner, J.D. Wadsworth, and J. 

Collinge. 2004. Characterization of two distinct prion strains derived from bovine spongiform 

encephalopathy transmissions to inbred mice. J. Gen. Virol. 8: 2471-2478. 

Mantze, U., G. Schlaf, R. Poethke, K. Felgenhauer, and M. Mäder. 1996. On the removal of nervous 

proteins from material used for gelatin manufacturing during processing. Pharm. Ind. 58: 837-841. 

Middleton, D., and R. Barlow. 1993. Failure to transmit bovine spongiform encephalopathy to 

mice by feeding them with extraneural tissues of affected cattle. Vet. Rec. 132: 545-547. 

Ministry of Agriculture, Fisheries, and Food. 1997. Food Safety Information Bulletin No. 85: Maternal 

Transmission. June. (http://archive.food.gov.uk/maff/archive/food/ 

bulletin/1997/no85/seac.htm) 

Ministry of Agriculture, Forestry, and Fisheries. 2003. BSE Epidemiological Study Group: Investigation 

into infection sources and infection routes of bovine spongiform encephalopathy (BSE). 

OIE. 2003. OIE expert ad hoc group reviews “atypical” BSE cases reported by Japan and Italy. 

December. Accessed September 7, 2007. (http://www.oie.int/eng/press/en_031208.htm) 

OIE. 2006. OIE official ‘disease-free’ recognition procedures. (www.oie.int/eng/info/ 

en_procedures.htm) 

OIE. 2009. Terrestrial Animal Health Code Chapter 11.6: Bovine Spongiform Encephalopathy. 

(www.oie.int/eng/normes/Mcode/en_chapitre_1.11.6.htm) 

OIE. 2009b. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 

(www.oie.int/Eng/Normes/Mmanual/A_summry.htm) 

OIE TAHSC. 2006. Supporting document for Chapter 2.3.13 of the Terrestrial Animal Health Code on 

Bovine Spongiform Encephalopathy. TAHSC Report. October. (www.aataanimaltransport.org/Publications/issues/4-GS75TAHSCBSEsupportdocOct2006.doc) 

Paisley, L., and J. Hostrup-Pedersen. 2004. Assessment of the risk of BSE transmission by tallow based 

calf milk replacer. Prev. Vet. Med. 63(1-2): 135 -149. 
33 

Okada H, Iwamaru Y, Imamura M, Masujin K, Matsuura Y, Murayama Y, Mohri S and Yokoyama T, 

2011. Detection of Disease-Associated Prion Protein in the Posterior Portion of the Small Intestine 

Involving the Continuous Peyer’s Patch in Cattle Orally Infected with Bovine Spongiform 

Encephalopathy Agent. Transboundary and Emerging Diseases. 

Okada H, Iwamaru Y, Imamura M, Masujin K, Yokoyama T and Mohri S, 2010. 

Immunohistochemical detection of disease-associated prion protein in the intestine of cattle 

naturally affected with bovine spongiform encephalopathy by using an alkaline-based chemical 

antigen retrieval method. J Vet Med Sci, 72, 11, 1423-1429. 

Prince, M.J., J.A. Bailey, P.R. Barrowman, K.J. Bishop, G.R. Campbell, and J.M. Wood. 2003. Bovine 

spongiform encephalopathy. Rev. Sci. Tech. 22(1): 37-60. 

Prusiner, S.B. 1994. Biology and Genetics of prion diseases. Annual review of Microbiology. 48: 655-

686. 

Schmidt, G.R., K.E. Hossner, R.S. Yemm, and D.H. Gould. 1999. Potential for disruption of central 

nervous system tissue in beef cattle by different types of captive bolt stunners. J. Food Prot. 62:390-393. 

SEAC. 2001. Minutes of the 73rd meeting held on 10 April 2002 at DEFRA. Accessed December 20, 

2007. (www.seac.gov.uk/papers/mins10-04-02.pdf) 

Smith, P., and R. Bradley. 2003. Bovine spongiform encephalopathy (BSE) and its epidemiology. British 

Medical Bulletin 66(1): 185-198. 

Taylor, D.M., S. L. Woodgate, and M. J. Atkinson. 1995. Inactivation of the bovine 

spongiform encephalopathy agent by rendering procedures. Vet. Rec. 137: 605-610. 

Taylor, D.M., S. L. Woodgate, A. J. Fleetwood, and R.J.G. Cawthorne. 1997. The effect of rendering 

procedures on scrapie agent. Vet. Rec. 141: 643-649. 

Taylor, D.M. 2000. Inactivation of transmissible degenerative encephalopathy agents: a review. Vet. J. 

159: 10-17. 

Taylor, D.M., and S.L. Woodgate. 2003. Rendering practices and inactivation of transmissible 

spongiform encephalopathy agents. Rev. Sci. Tech. 22(1): 297-310. 

Terry, L.A., S. Marsh, S.J. Ryder, S.A.C. Hawkins, G.A.H. Wells, and Y.I. Spencer. 2003. Detection of 

disease specific PrP in the distal ileum of cattle exposed orally to the 

agent of bovine spongiform encephalopathy. Vet. Rec. 152: 387-392. 

Tsutsui, T., T. Yamamoto, S. Hashimoto, T. Nonaka, A. Nishiguchi, and S. Kobayashi. 2008. Letter to 

the editor: Milk replacers and bovine spongiform encephalopathy in calves, Japan. Emerg. Infect. Dis. 

14(3): 525-526. 

USDA. 2006. Bovine spongiform encephalopathy (BSE) ongoing surveillance plan. 

(www.aphis.usda.gov/newsroom/hot_issues/bse/downloads/BSE_ongoing_surv_plan_final_ 

71406%20.pdf) 

Wells, G.A.H., M. Dawson, S.A.C. Hawkins, R.B. Green, I. Dexter, M.E. Francis, M.M. 
34 

Simmons, A.R. Austin, and M.W. Horigan. 1994. Infectivity in the ileum of cattle challenged orally with 

bovine spongiform encephalopathy. Vet. Rec. 135: 40-41. 

Wells, G.A.H., M. Dawson, S.A.C. Hawkins, A.R. Austin, R.B. Green, I. Dexter, M.W. 

Horigan, and M.M. Simmons. 1996. Preliminary observations on the pathogenesis of 

experimental bovine spongiform encephalopathy. In: Bovine spongiform encephalopathy: 

the BSE dilemma (Gibbs C.J. Jr., ed.). New York: Springer-Verlag. 

Wells, G.A.H., S.A.C. Hawkins, R.B. Green, A.R. Austin, I. Dexter, Y.I. Spencer, M.J. 

Chaplin, M.J. Stack, and M. Dawson. 1998. Preliminary observations on the pathogenesis of experimental 

bovine spongiform encephalopathy (BSE): an update. Vet. Rec. 142: 103-106. 

Wells, G.A.H., S.A.C. Hawkins, R.B. Green, Y.I. Spencer, I. Dexter, and M. Dawson. 

1999. Limited detection of sternal bone marrow infectivity in the clinical phase of 

experimental bovine spongiform encephalopathy (BSE). Vet. Rec. 144: 292-294. 

Wells, G.A.H., J. Spiropoulos, S.A.C. Hawkins, S.J. Ryder. 2005. Pathogenesis of experimental bovine 

spongiform encephalopathy: preclinical infectivity in tonsil and observations on the distribution of lingual 

tonsil in slaughtered cattle. Vet. Rec. 156(13): 401-407. 

Wells, G.A.H. 2008. Personal communication with APHIS. E-mail, January 30, 2008. 

Wilesmith, J.W., G.A.H. Wells, M.P. Cranwell, and J.B.M. Ryan. 1988. Bovine spongiform 

encephalopathy epidemiological studies. Vet. Rec. 123: 638-644. 

Wilesmith, J.W., J.B.M. Ryan, and M.J. Atkinson. 1991. Bovine spongiform encephalopathy: 

epidemiological studies of the origin. Vet. Rec. 128: 199-203. 

Wilesmith, J.W., J.B.M. Ryan, and W.D. Hueston. 1992. Bovine spongiform encephalopathy: casecontrol studies of calf feeding practices and meat-and-bone meal inclusion in proprietary concentrates. 

Res. Vet. Sci. 52: 325-331. 

Wilesmith, J.W., G.A.H. Wells, J.B.M. Ryan, D. Gavier-Widen, and M.M. Simmons. 1997. A cohort 

study to examine maternally-associated risk factors for BSE. Vet. Rec. 141: 239-243. 

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 encephalopathy (BSE). Vet. Rec. 150(12): 365-378. 

Yamakawa, Y., K. Hagiwara, K. Nohtomi, Y. Nakamura, M. Nishijima, Y. Higuchi, Y. Sato, T. Sata, and 

the Expert Committee for BSE Diagnosis, 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 steer. 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 distribution, 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 (Prince et al. 2003; Wilesmith et al. 1988, 1991, 1992). 

Contamination occurs when abnormal proteins derived from specific tissues originating from 

infected ruminants are incorporated into feed. Standard rendering processes do not completely 

inactivate the BSE agent. Therefore, 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 mitigate the risk of BSE transmission (OIE, 2009). 

Early studies suggested that maternal transmission 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, however, 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 last 6 months of incubation of the disease in its dam (Donnelly et 

al. 2002). More recent work on cases born after the 1996 feed ban fails to demonstrate evidence 

of maternal transmission (Hill 2005). Thus, although 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 modelling have supported the view that most cases 

were likely exposed as calves (Wilesmith et. al 1988) with the greatest chance of becoming 

infected up to or around the first year of life (Wilesmith et al. 1992, 1992a; Ferguson 1997; De 

Koeijer et al. 2004; Arnold and Wilesmith 2004). These findings suggest that susceptibility in 

cattle declines with age, and therefore young animals are most susceptible. One mathematical 

model assumes that susceptibility declines exponentially after the age of 4 months, levelling off 

at 10 percent of the peak value (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 replacement dairy cattle and BSEepidemic data to back-calculate the likely age of infection. This study found that dairy cattle 

were at greatest risk in their first 6 months. Although this result 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 implementation of a ruminant-to-ruminant feed ban 

exerts downward pressure on the prevalence of BSE (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 specified bovine offals was banned for use in any 

animal feed in the UK. This ban prohibited the use in any animal feed of bovine tissues with the 

highest potential concentration 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 meat-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 (Department 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 presented in the following figure and table represent the specific 

situation in the UK1

 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 for 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 Man. Records published elsewhere indicate that the vast majority of BSE cases in the UK

have been detected in Great Britain (OIE 2008). Therefore, epidemiologic 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 

Implementation 
Appendix: 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 ban was reinforced in 1996 (BAB cases). Several 

analyses of the BAB cases suggest that the cattle were likely to have been exposed to an 

exogenous feed source from continental Europe. For instance, an analysis of the first 16 cases 

(Wilesmith 2002) indicates that an exogenous (non-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 ban 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 has occurred in a number of other BSE-affected 

countries. A recent report (Hill 2005) also describes 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 cases have been born in 2002 (UK, Portugal and 

Spain) and one in 2003 (Poland), which tested positive at only 25 months of age (EFSA 2007). 

Incubation 

BSE has a prolonged incubation period. Epidemiological data from the UK epidemic have 

demonstrated that, on average, cattle develop clinical signs 4 to 6 years after infection (Bradley 

1991; Anderson et al. 1996). In BSE, as in other TSEs, the total amount of infectivity in an 
Appendix: 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 animal. Infectivity is considered to increase exponentially after 

exposure, reaching 3 logs less than clinical cases 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 clinical signs of disease become apparent). The Department for 

Environment Food and Rural Affairs (DEFRA) UK has carried out two oral challenge studies in 

cattle to determine the incubation period, inoculating varying amounts of brainstems pooled from 

BSE-affected cattle (Wells et al. 2007). In the first 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 animals 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 pathological evidence of disease. 

The second attack rate experiments 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 the 0.1-g group (IP range 53–98 months), 1 out of 

15 in the 0.01-g group (IP range 56 months), and 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 incubation period for 1 g dose is 45 months and the 

mean is 60 months, which suggests that the infectivity in CNS-related SRM would most likely be 

absent in cattle aged 35 months. 

The UK data show that of the cattle that developed clinical BSE in the field, only 0.01 percent 

were less than 30 months of age (EC SSC 2002). Thus, cattle less than 30 months of age are less 

likely to be in the later stages of BSE incubation than older BSE-infected cattle and hence are 

less likely to contain high levels of BSE infectivity. In addition, research demonstrates that the 

incubation period for BSE appears to be linked to the infectious dose of the BSE agent received, 

i.e., the larger the infectious dose received, the shorter the 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 BSE agent at a very young age. 

Tissue distribution and infectivity 

Most of the information on the development and distribution of tissue infectivity in BSE-infected 

cattle has been derived from experimental pathogenesis studies conducted in the UK (Wells et al. 

1994, 1998, 1999). In these studies, cattle were deliberately infected with BSE through oral 

exposure to the brain tissue of cattle with confirmed BSE. Subsets of the experimentally infected 
Appendix: Characteristics of the BSE Agent 

39 

cattle were killed at regular intervals as the disease progressed. At each interval, the tissues of the 

infected cattle were examined for histopathological changes consistent with BSE and for 

abnormal prion proteins. Also, at each 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 containing 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 (Wells et al. 1994, 1998, 1999). This dose is probably 

10 to 100 times greater than that associated with field exposure via feed (DEFRA 2006). The 

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

as well as the distribution of infectivity in the animal’s body, changes over time (Wells et al. 

1994, 1998, 1999). First, infectivity 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 found in cattle with 

clinical signs of BSE in the brain, spinal cord, dorsal root ganglia, trigeminal ganglia, and the 

distal ileum of the small intestine. 

A different study (Espinosa et al. 2007), utilizing material derived from the UK pathogenesis 

study (100-g dose of BSE-infected brainstem materials orally inoculated into cattle), reported the 

detection of infectivity (by assay in transgenic BoPrP-Tg110 mice genetically altered to be 

highly susceptible to BSE) in the brainstem of cattle killed 27 months after exposure. A different 

study (Masujin et al. 2007) detected the presence of PrPsc in the vagus nerve and adrenal gland 

obtained from clinically confirmed BSE cases 

BSE infectivity was demonstrated in the brain, spinal cord, and dorsal root ganglia as early as 32 

months after oral exposure to the BSE agent in some cattle (Wells et al. 1994, 1998, 1999). 

Infectivity was demonstrated in these tissues 3 months 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 and again at 38 months and 40 months after oral 

exposure. Regarding the distal ileum, studies examining the distribution of PrPSc by 

immunohistochemistry of experimentally infected cattle confirmed that PrPSc 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 PrPSc by immunostaining in the duodenum, jejunum, 

cecum, colon, myenteric plexus, and submucosal plexus of calves orally exposed to large doses 

of the BSE agent and killed 6 months after exposure. In contrast, immunostaining of follicles 

from Peyer’s patches of the distal ileum did reveal the presence of PrPSc in these same calves. 

The distal ilea of 29 naturally occurring cases of BSE were examined for PrP by 

immunohistochemistry. Sparse PrPSc-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 challenge 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 immunostaining in the submucosal 

plexus of the distal ileum of any of the infected cattle was observed, suggesting a very low level 

of infectivity in the enteric 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 the distal ileum, moderate amount in ileocaecal 

junction and traces of infectivity in the jejunum. The higher amounts of infectivity were mainly 

seen in younger cattle (8 to 12 months post inoculation). Recent examination of intestinal 

samples obtained from the UK pathogenesis study (Stark, 2011) using the mouse bioassay 

found immunostaining of the intestinal nervous system was rare. Since the enteric nervous 

system extends throughout the small intestine, the 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, which used a cattle bioassay as an endpoint, was 

conducted to ensure that low levels of infectivity that may not have been detected in the first 

phase using the mouse bioassay were not missed (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 bioassay 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 agent 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-inoculation, the level of infectivity 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 ID50 per gram of tissue 

as determined by endpoint titration in Tgbov 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 Western 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 (Hoffman et al, 2011) found high amounts of 

infectivity in the distal ileum, moderate amount in ileocaecal junction, and traces of infectivity in 

the jejunum. 

The amount and distribution of infectivity in specific tissues from an infected cow have been 

estimated by Comer and Huntley (2003) in their evaluation of the available literature. Those 

summary results, presented in table 2, describe distribution of infectivity in various tissues, 

including brain, spinal cord, dorsal 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 of 
Appendix: Characteristics of the BSE Agent 

41 

estimated cattle oral infectious dose-50 (ID50) units3 per gram, and the total number of cattle oral 

ID50 units attributed to each tissue to estimate a percentage of cattle oral ID50 units for each 

tissue.

3 BSE infectivity is expressed in terms of cattle oral infectious dose-50 units (ID50). A cattle ID50 is defined as the amount of 

infectivity required to cause infection in 50 percent 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 ID50)

Tissue Weight 

(g/animal)

Infectivity % ID50/g ID50/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 clinically affected cattle is associated 

with central and peripheral nervous system tissues, i.e., brain, spinal cord, dorsal root ganglia, 

and trigeminal ganglia. About 10 percent was associated with the distal ileum. Minimal 

infectivity was associated with tonsils in a clinically affected animal. 

Bones 

Early studies of cattle challenged orally with 100 g of BSE infected 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 detected, using the mouse bioassay, in the sternal bone marrow from 

pooled samples of animals killed in the clinical phase of the disease at 38 months post 

inoculation but not before and 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 signs 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 sensitivity of the oral exposure study, additional assays of selected tissues were 

inoculated intracerebrally in cattle. These studies are approximately 500-fold more sensitive than 

the standard mouse bioassay. Bone marrow samples 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 larger pathogenesis study in Germany did not 

demonstrate PrPsc in the bone marrow (femur) of two clinically 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 incorporated the use of transgenic mice designed to 

overexpress bovine prion protein as a way to improve 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 (Buschmann and Groschup 2005). The sensitivity of these 

mice to infection was shown to be significantly greater than other mice panels used in bioassays. 

In addition, the sensitivity was shown to be even greater than that of cattle by approximately 

tenfold. This study demonstrated low levels of infectivity in the facial and 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 also detected in the vagus nerve, adrenal gland, 

and weakly from the sciatic nerve from clinically confirmed BSE cases (Masujin 2007). 

While these are findings that can help further characterize the pathogenesis of BSE, they cannot 

be easily extrapolated into the context of the risk presented by natural exposure pathways. The 

findings may be influenced by the overexpression of prion proteins in these genetically 

engineered mice. Any apparent levels of infectivity are low and would be even lower in other 

species such as cattle. Given all of these factors, we conclude that there is not sufficient 

information in this study to alter our understanding of the epidemiologically significant 

distribution of BSE infectivity in cattle. 

Appendix References 

Anderson, R.M., C.A. Donnelly, N.M. Ferguson, M.E. Woolhouse, C.J. Watt, H.J. Udy, S. MaWhinney, 

S.P. Dunstan, T.R. Southwood, J.W. Wilesmith, J.B. Ryan, L.J. Hoinville, J.E. Hillerton, A.R. Austin, 

and G.A. Wells. 1996. Transmission dynamics and epidemiology of BSE in British cattle. Nature 

382(6594): 779-788. 

Arnold, M., and J. Wilesmith. 2004. Estimation of the Age-Dependent BSE Infection of Dairy Animals in 

Great Britain. Prev. Vet. Med. 66: 35-47. 

Bradley, R. 1991. Bovine spongiform encephalopathy (BSE): the current situation and 

research. Eur. J. Epidemiol. 7: 532-544. 

Buschmann, A., and M. Groschup. 2005. Highly bovine spongiform encephalopathysensitive transgenic mice confirm the essential restriction of infectivity to the nervous 

system in clinically diseased cattle. J. Infect. Dis. 192: 934-942. 

Cohen, J., K. Duggar, G. Gray, S. Kreindel, H. Abdelrahman, T. Habtemariam, D. Oryang, and B. 

Tameru. 2001. Evaluation of the potential for bovine spongiform encephalopathy in the United States. 

http://www.aphis.usda.gov/newsroom/hot_issues/bse/background/documents/mainreporttext.pdf 

Comer, P.J. and P.J. Huntly. 2003. Exposure of the human population to BSE 
Appendix: Characteristics of the BSE Agent 

44 

infectivity over the course of the BSE epidemic in Great Britain and the impact of 

changes to the over thirty month rule. Over Thirty Month Rule (OTMR) review paper. 

June. Accessed September 7, 2007. (http://www.food.gov.uk/multimedia/pdfs/otmcomer.pdf) 

De Koeijer, A., H. Heesterbeek, B. Schreuder, R. Oberthur, J. Wilesmith, H. van Roermund, and M. de 

Jong. 2004. Quantifying BSE control by calculating the basic reproduction ratio R0 for the infection 

among cattle. J. Math. Biol. 48(1): 1-22. 

DEFRA, United Kingdom. 2006. BSE: Science and research: pathogenesis. Page last reviewed October 

10. (http://www.defra.gov.uk/animalh/bse/science-research/pathog.html) 

DEFRA, United Kingdom. 2008. BSE: Statistics – Monthly Statistics (as of January 2, 2008). 

(www.defra.gov.uk/animalh/bse/statistics/pdf/monthly.pdf) 

Donnelly, C.A., N.M. Ferguson, A.C. Ghani and R.M. Anderson. 2002. Implications of BSE infection 

screening data for the scale of the British BSE epidemic and current European infection levels. Proc. Biol. 

Sci. 269(1506): 2179-2190. 

Espinosa, J.C., M. Morales, J. Castilla, M. Rogers, and J.M. Torres. 2007. Progression of prion infectivity 

in asymptomatic cattle after oral bovine spongiform encephalopathy challenge. J. Gen. Virol. 88: 1379-

1383. 

EC SSC. 2002. Update of the opinion on TSE infectivity distribution in ruminant tissues. November. 

(http://www.europa.eu.int/comm/food/fs/sc/ssc/out296_en.pdf) 

EFSA. 2007. Opinion of the scientific panel on biological hazards. EFSA Journal 476: 1-47. 

Ferguson, N.M., C.A. Donnelly, M.E.J. Woolhouse, and R.M. Anderson. 1997. The epidemiology of BSE 

in cattle herds in Great Britain 2. Model construction and analysis of transmission dynamics, Phil. Trans. 

R. Soc. B 352 (1355). pp. 803–838. 

Hill, W. 2005. Review of the evidence for the occurrence of ‘BARB’ BSE cases in cattle. July 5. 

Accessed September 7, 2007. (http://www.defra.gov.uk/animalh/bse/pdf/hillreport.pdf) 

Hoffmann, C., U. Ziegler, A. Buschmann, A. Weber, L. Kupfer, A. Oelschlegel, B. Hammerschmidt, and 

M. Groschup. 2007. Prions spread via the autonomic nervous system from the gut to the central nervous 

system in cattle incubating bovine spongiform encephalopathy. J. Gen. Virol. 88: 1048-1055. 

Hoffmann C, Eiden M, Kaatz M, Keller M, Ziegler U, Rogers R, Hills B, Balkema-Buschmann A, van 

Keulen L, Jacobs JG, Groschup MH. BSE infectivity in jejunum, ileum and ileocaecal junction of 

incubating cattle. Vet Res. 2011 Feb 7;42(1):21. 

Masujin, K., D. Matthews, G.A.H. Wells, S. Mohri, and T. Yokoyama. 2007. Prions in the peripheral 

nerves of bovine spongiform encephalopathy-affected cattle. J. Gen. Virol. 88: 1850-1858. 

Prince, M.J., J.A. Bailey, P.R. Barrowman, K.J. Bishop, G.R. Campbell, and J.M. Wood. 2003. Bovine 

Spongiform Encephalopathy. Rev. Sci. Tech. 1: 37-60. 

Stack MJ, Moore SJ, Vidal-Diez A, Arnold ME, Jones EM, Spencer YI, Webb P, Spiropoulos J, 

Powell L, Bellerby P, Thurston L, Cooper J, Chaplin MJ, Davis LA, Everitt S, Focosi-Snyman R, 
Appendix: Characteristics of the BSE Agent 

45 

Hawkins SAC, Simmons MM and Wells GAH, 2011. Experimental bovine spongiform 

encephalopathy: detection of PrPSc in the small intestine relative to exposure dose and age. 

Journal of Comparative Pathology 

Terry, L. A., S. Marsh, S.J. Ryder, S.A.C. Hawkins, G.A.H. Wells, and Y.I. Spencer. 2003. Detection of 

disease specific PrP in the distal ileum of cattle exposed orally to the agent of bovine spongiform 

encephalopathy. Vet. Rec. 152: 387-392. 

Wells, G.A.H., M. Dawson, S.A.C. Hawkins, R.B. Green, I. Dexter, M.E. Francis, M.M. Simmons, A.R. 

Austin, and M.W. Horigan. 1994. Infectivity in the ileum of cattle challenged orally with bovine 

spongiform encephalopathy. Vet. Rec. 135: 40-41. 

Wells, G.A.H., S.A.C. Hawkins, R.B. Green, A.R. Austin, I. Dexter, Y.I. Spencer, M.J. Chaplin, M.J. 

Stack, and M. Dawson. 1998. Preliminary observations on the pathogenesis of experimental bovine 

spongiform encephalopathy (BSE): an update. Vet. Rec. 142: 103-106. 

Wells, G.A.H., S.A.C. Hawkins, R.B. Green, Y.I. Spencer, I. Dexter, and M. Dawson. 1999. Limited 

detection of sternal bone marrow infectivity in the clinical phase of experimental bovine spongiform 

encephalopathy (BSE). Vet. Rec. 144: 292-294. 

Wells, G.A.H., J. Spiropoulos, S.A.C. Hawkins, S.J. Ryder. 2005. Pathogenesis of experimental bovine 

spongiform encephalopathy: preclinical infectivity in tonsil and observations on the distribution of lingual 

tonsil in slaughtered cattle. Vet Rec. 156(13):401-407. 

Wells, G.A.H., T. Konold, M.E. Arnold, A.R. Austin, S.A.C. Hawkins, M. Stack, M.M. Simmons, Y.H. 

Lee, D. Gavier-Widén, M. Dawson, and J.W. Wilesmith. 2007. Bovine spongiform encephalopathy: the 

effect of oral exposure dose on attack rate and incubation period in cattle. J. Gen. Virol. 88: 1363-1373. 

Wells, G.A.H. 2008. Personal communication with APHIS. E-mail, January 30, 2008. 

Wilesmith, J.W., G.A.H. Wells, M.P. Cranwell, and J.B.M. Ryan. 1988. Bovine spongiform 

encephalopathy epidemiological studies. Vet. Rec. 123: 638-644. 

Wilesmith, J.W., J.B.M. Ryan , and M.J. Atkinson. 1991. Bovine spongiform encephalopathy: 

epidemiological studies of the origin. Vet. Rec. 128: 199-203. 

Wilesmith, J.W., J.B.M. Ryan, and W.D. Hueston. 1992. Bovine spongiform encephalopathy: casecontrol studies of calf feeding practices and meat-and-bone meal inclusion in proprietary concentrates. 

Res. Vet. Sci. 52: 325-331. 

Wilesmith J.W., J.B.M. Ryan, W.D. Hueston, and L.J. Hoinville. 1992a. Bovine spongiform 

encephalopathy: epidemiological features 1985 to 1990. Vet. Rec. 130(5): 90-94. 

Wilesmith, J.W., G.A.H. Wells, J.B.M. Ryan, D. Gavier-Widen, and M.M. Simmons. 1997. A cohort 

study to examine maternally-associated risk factors for BSE. Vet. Rec. 141: 239-43. 

Wilesmith, J.W. 2002. Preliminary epidemiological analyses of the first 16 cases of BSE born after July 

23, 1996 in Great Britain. Vet. Rec. 151: 451-452.