bovine spongiform encephalopathy

[Note that the ‘Host Animals’ table above relates to classical BSE]

c-BSE

Classical BSE (c-BSE) was first recognized in 1984-85 as a novel prion disease affecting cattle in the UK (Wells et al., 1987). The origin of c-BSE has still not been clearly established, but the number of cases was amplified by the recycling of infected carcasses into cattle feed in the form of meat and bone meal (MBM) (Wilesmith et al., 1988).

In the 1990s, c-BSE cases were observed in captive animals belonging to a variety of species such as kudu, nyala, Arabian onynx, Scimitar horned oryx, eland, gemsbok, bison, tiger, cheetah, ocelot and puma. c-BSE transmission was also observed in domestic cats. These cases were the probable consequence of a dietary exposure to c-BSE contaminated feed material (processed animal proteins or c-BSE infected carcasses) (Collinge, 2001).

The expansion of the c-BSE epizootic led to the consideration that this disease was a potential threat to small ruminants. To date, only two natural c-BSE cases have been officially identified in farmed small ruminants. Both cases occurred in goats. The first one was detected in 2002 by the active surveillance system in France (Eloit et al., 2005). The second case was initially reported as a scrapie case in 1990 in the UK but confirmed to be a c-BSE case in 2011 (Spiropoulos et al., 2011).

Successful experimental transmission of c-BSE has been reported in a large number of host species: cattle, pig (transmission by the intracerebral route but not by the oral route), sheep, goats, cervids, various rodent species, mink and primates (Dagleish et al., 2008; Hedman et al., 2018; Houston and Andreoletti, 2019). These observations indicate the high capacity of c-BSE to cross the species barrier that naturally limits the capacity of TSE to propagate between species.

Exposure of fish (Sparus aurata, guilt head bream, by the oral route) or chicken (by the intracerebral route) to the c-BSE agent failed to cause transmission of the disease or the accumulation of detectable levels of PrP^Sc or prion infectivity (Salta et al., 2009; Moore et al., 2011).

Atypical BSEs

H-BSE- and L-BSE have only been identified in domestic cattle. Both L-BSE and H-BSE are transmissible by the intracerebral route to cattle (Balkema-Buschmann et al., 2011b, Suardi et al., 2012; Konold et al., 2014; Vallino Costassa et al., 2018). Oral transmission of L-BSE to cattle was also demonstrated (Okada et al., 2017a). The abilities of L- and H-BSE to propagate in farmed animal species remains to be established. So far, L-BSE has been successfully transmitted to mice, sheep, bank voles, primates and hamsters by the intracerebral route. H-BSE has not yet proven transmissible to sheep or primates (Comoy et al., 2008; Nicot et al., 2014; Gielbert et al., 2018).

c-BSE

In the UK, after its identification in 1985, the c-BSE epizootic peaked in 1992 with about 180,000 clinical cases in total (Wells et al., 1987). c-BSE was disseminated to at least 28 countries (including the USA, Canada, Japan and countries in Europe) through the export of infected live animals and/or contaminated MBM and livestock feed. After the UK, the European Union was the second most c-BSE-affected geographical area with a total of 6,193 cases recorded between 1989 and 2016 (European Food Safety Authority, 2019).

The OIE provides the list of BSE cases reported by its member states and an updated map of countries according to their c-BSE risk status.

Atypical BSE cases

L-BSE cases were reported in France, Italy, Germany, Great Britain, Romania and Poland. H-BSE has been reported in France, Great Britain, Germany, the Netherlands, Norway and Sweden. Outside the EU, atypical BSE has also been reported in Japan, the USA, Canada, Brazil and Switzerland (in a zebu). Interestingly, atypical BSE cases were identified in countries where no c-BSE cases have been observed (such as Brazil and Norway) (European Food Safety Authority, 2019). Considering the characteristics of atypical BSEs (low prevalence, cases detected in aged animals by active surveillance), the detection of these conditions in cattle populations remains difficult and the final geographical distribution of these two forms of bovine prion disease remains uncertain.

Clinical Diagnosis

c-BSE

Under natural conditions, clinical c-BSE cases have been observed in animals aged between 20 months and 22 years old. In the UK, during the early phase of the c-BSE epizootic (1986 to 1996) most cases occurred in cattle aged between 3 and 5 years old but over the following 15 years, the average age of cattle with confirmed signs of disease has gradually increased to over 12 years (European Food Safety Authority, 2019). This evolution was likely a consequence of a decline in the level of exposure of cattle to the c-BSE agent. End-point oral titration in cattle with decreasing amounts of c-BSE-affected cattle brain homogenate confirmed the progressive increase of the incubation period. In cattle that received very low amounts of infectious material (1 mg of brain equivalent material), disease was observed in some animals after more than 75 months of incubation (Arnold et al., 2009).

In natural c-BSE cases, early clinical signs include changes in anxiety/aggressiveness, and discrete gait/locomotion disturbance. As the disease progresses, the animals display increasing hyperaesthesia (to light, sound and touch), and stimulus-induced myoclonic jerks (to touch and sound), ataxia and loss of weight. The duration of the clinical phase can range from a few weeks to over 6 months (Konold et al., 2004). A video illustrating symptoms that can be found in c-BSE affected cattle can be found on the website of the APHA, Weybridge, UK, the former European TSE Reference Laboratory (https://science.vla.gov.uk/tseglobalnet/training.html)

Atypical-BSEs

To date, no natural cases of H- or L-type BSE have been identified on the basis of their clinical signs. The available clinical data for these types of BSE are all derived from experimental transmission in cattle inoculated by the intracerebral route. These data might inaccurately reflect the course/symptoms of natural disease.

In experimental L-BSE cattle inoculated by the intracerebral route, early clinical signs involved muscular fasciculation (of the hind limbs), dullness and postural changes (such as low head carriage and mild kyphosis). As the disease progressed, muscle atrophy starting in the gluteal area and progressing to the paravertebral region became apparent. Hyper-reactivity to facial touch was also commonly observed. Progressive ataxia associated with difficulties in rising appeared as the disease progressed. The duration of the clinical phase in L-BSE challenged cattle was on average 2 months (10 and 105 days, n=2) (Konold et al., 2012).

In experimental H-BSE cases, symptoms and clinical phase duration (on average 2 months) were very similar to those observed in L-BSE experimental cases. The first clinical sign was a loss of weight that led to a deteriorating body condition. Animals displayed dullness and non-physiologically low head positions. H-BSE inoculated cattle showed hyper-responsiveness to acoustic and visual stimuli, and to tactile stimuli on their heads. As the disease progressed, the animals developed a progressive hind-limb ataxia that led rapidly to decumbency (downer-cow syndrome) (Konold et al., 2012).

Although some differences might be observed between the clinical signs displayed in experimental cases of atypical BSE and classical BSE in cattle, it is not possible to differentiate between the three forms of BSE using clinical presentation alone.

Differential Diagnosis

Non-infectious differential diagnosis should include the nervous form of ketosis, lead toxicity, CNS tumours and hypomagnesaemia. Infectious differential diagnoses should include listeriosis, rabies and Aujeszky’s disease.

Laboratory Diagnosis

Currently, there is no method capable of confirming the presence of any type of BSE in live cattle.

All post mortem samples intended to be examined for the presence of TSE shall in general be collected using the methods and guidelines laid down in the latest edition of the Manual for Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organisation for Animal Health (OIE).

Histopathology

Historically, c-BSE confirmation in clinically suspect animals (identified by passive surveillance) has relied on the identification of vacuolar changes in the brain by histopathologic examination of formalin-fixed/paraffin embedded brain sections (Wells et al., 1989). The discovery of the central role of PrP^Sc in prion diseases followed by the development of antibodies against the prion protein that allowed immuno-detection of the Proteinase K (PK)-resistant moiety of the PrP^Sc opened the way for the development of various methodologies for c-BSE diagnosis (Merz et al., 1981; Merz et al., 1983; Somerville et al., 1986; Grassi et al., 2000). Vacuolar changes appear at a very late stage of the incubation period in c-BSE infected cattle. In contrast, PrP^Sc accumulation can be detected in the brains of c-BSE infected animals several months before clinical onset (Arnold et al., 2009). As a consequence, PrP^Sc immuno-detection is now the standard approach applied in BSE surveillance and confirmatory testing in suspected animals.

Detection of disease-associated PrP by immunohistochemistry

PrP^Sc detection in the brain by immunohistochemistry (IHC) is one of the two main confirmatory methods that are used for BSE diagnosis in cattle. IHC is performed on the same material (formalin-fixed paraffin-embedded tissue) that is used for the conventional histopathological examination. The combination of vacuolar changes and PrP^Sc deposition in certain neuroanatomical areas of the bovine brain provides a high level of reliability for c-BSE diagnosis. When performing IHC for disease-associated PrP detection a pre-treatment of the slides is necessary to reduce/eliminate PrP^C and enhance PrP^Sc immunoreactivity. Numerous PrP^Sc IHC detection protocols are available in the literature. Their performances can vary significantly according to the protocol for slide pre-treatment and the nature of the anti-PrP antibody. PrP^Sc detection by IHC can be performed on formalin-fixed autolysed tissues (Monleón et al., 2003). However, when autolysis precludes the recognition of the anatomical structures in the brain, the reliability of the IHC diagnosis can be affected and, in this context, use of the western blot technique is suitable for BSE diagnosis.

Detection of disease-associated PrP by western blot

Western blot (WB) detection of PK-digested disease-associated PrP constitutes the second main reference method for the confirmation of BSE diagnosis. This method is highly specific and reliable. The SAF-immunoblot was the first WB protocol used for c-BSE diagnosis. This protocol requires a large amount of starting material (2-4 g) and is labour/time consuming due to the requirement of precipitation of disease-associated PrP by ultracentrifugation. Modern PrP^Sc extraction methods require lower amounts of brain material (about 100-fold less) than SAF immunoblots and only 2 hours of benchwork. Detection of PK-resistant PrP^Sc by WB can now be executed in less than 6 hours. WB can be applied to autolysed tissues without impairing the specificity/sensitivity of PrP^Sc detection (Hayashi et al., 2004).

The analytical sensitivity of different PK-resistant PrP^Sc WB detection protocols varies significantly and is highly dependent upon the extraction method used to prepare disease-associated PrP and the particular anti-PrP antibody used (Morel et al., 2004; Féraudet et al., 2005). Periodic ring trial testing of a common set of reference samples plays a key role in maintaining the quality and consistency of BSE detection by WB in reference laboratories. WB is the most straightforward approach to discriminate between H-BSE, L-BSE and c-BSE cases. Discrimination is based on distinct N-terminal PK cleavage, antibody binding and glycosylation pattern of PK-resistant PrP^Sc (Jacobs et al., 2007).

Rapid PrPSc detection assays

The c-BSE epizootic stimulated development of commercial kits for the detection of disease-associated PrP for the diagnosis of c-BSE. These so-called rapid tests were designed with the objective of an accelerated test time (diagnostic results in less than 24h) and large-scale screening of healthy slaughtered and rendered cattle (Grassi et al., 2008). These rapid tests rely on the detection of disease-associated PrP by different immuno-detection methodologies such as WB, ELISA or immuno-chromatographic assays.

The European union established a standard procedure to validate and periodically assess the performances of rapid tests proposed for the detection of BSE. The EU approval process has been accepted as the gold standard by the OIE. However, certain countries, such as the USA, Canada and Japan, have established their own evaluation mechanisms. Since 2001, there has been a continual evolution of the list of cattle TSE rapid tests. At the time of writing, the following assays are approved and marketed for BSE testing in the European Union (European Food Safety Authority, 2019):

• Prionics-Check Western
• Bio-Rad TeSeE SAP
• Prionics-Check LIA
• IDEXX HerdChek BSE Antigen,
• IDEXX HerdChek BSE-Scrapie EIA Antigen
• Prionics-Check PrioSTRIP
• Roboscreen Beta Prion BSE EIA

The distribution and use of TSE rapid tests are restricted to government-approved laboratories in charge of prion disease surveillance programs. 

Following its appearance in the UK, c-BSE was disseminated to numerous countries by the import of live cattle and/or contaminated animal products and feed material. In reaction, the OIE produced recommendations aimed at preventing the entry of c-BSE in disease-free countries and assisted to progressively control and eradicate the disease in countries where c-BSE was present. Over the last 30 years these recommendations have evolved to adapt to the changes in our understanding and knowledge of bovine TSE diseases. Numerous countries, in particular those where c-BSE epizootics had severe adverse consequences, including EU nations and Canada, have implemented surveillance, control and eradication measures that exceed the OIE recommendations. As occurs with other bovine diseases, individual cattle identification enables effective surveillance and tracing of suspected animals and plays a central role in the surveillance, the control and the eradication of c-BSE. 

Prevention

In 2000 the OIE provided a methodology to assess the risk of c-BSE presence and circulation in countries/geographical areas (GBR: geographical BSE risk). The GBR methodology relies on the assessment of:

• The risk of entry of c-BSE into a territory by the import of ruminants or animal products
• The risk of cattle exposure to the c-BSE agent including rendering practices, production and use of processed animal proteins
• The existence and level of BSE surveillance in cattle populations
• Transparent and reliable reports of data on the BSE epidemiological situation

Based on these criteria the BSE risk in countries/geographical areas is regularly updated and classified as:

• Negligible c-BSE risk countries
• Controlled c-BSE risk countries
• Countries without an OIE official c-BSE status

The OIE risk status only addresses the risk of c-BSE as it is considered that atypical BSE corresponds to a spontaneous disease in cattle.

Negligible BSE risk countries are defined as countries:

• That have had a negligible risk with regards to c-BSE for at least 7 years
• Where an adequate surveillance program has been implemented and is maintained for at least 7 years
• Where no autochthonous c-BSE case has been observed in animals born less than 11 years ago
• Where a documented ruminant-to-ruminant feed ban has been in place for at least 8 years

The OIE BSE status provides a basis for national import/export regulations applicable to live cattle, certain commodities and animal products between areas with different BSE status (OIE terrestrial animal health code Article 11.4.6 to 11.4.20)

Monitoring and surveillance

For more than 20 years passive surveillance, which refers to the clinical identification and testing of c-BSE suspect cases, has remained the only available approach for the monitoring and surveillance of the TSE in ruminants. In the early 1990s passive surveillance allowed the identification of c-BSE cases outside the UK in countries such as France, Ireland and Portugal. However, the characterization of c-BSE pathogenesis in cattle rapidly led to the consensus that passive surveillance has a limited value for the detection and epidemiological monitoring of the disease. At the end of the 1990s the development of rapid tests (immuno-detection of diseases-associated PrP in the brainstem), which allow an earlier and more sensitive detection than passive surveillance, provided a paradigm shift in BSE diagnosis. From 1998 the European Commission instigated a harmonized BSE surveillance program within its member states and from 2000 rapid tests have been used to implement large scale and systematic BSE surveillance programs in cattle populations. Since the start of these programmes, approximately 100 million animals have been tested by rapid tests in the framework of the EU TSE surveillance programs.

Currently, the BSE active surveillance program relies on the screening of targeted categories of cattle using rapid tests in approved laboratories. In the case of an initial positive reaction, the sample is submitted for confirmatory testing at a reference laboratory. When a positive BSE diagnosis is established, a discriminatory assay (generally WB) is performed to identify whether the bovine TSE is c-BSE, H-BSE or L-BSE (European Food Safety Authority, 2019).

The EU TSE surveillance follows EU Regulation (EC) 999/2001. This regulation was amended several times and from 2009, the age categories of the cattle to be tested were progressively narrowed as shown by the surveillance by period (year and age of cattle) in table 1 below. This evolution of the surveillance by period reflected the c-BSE epidemiological situation in the EU member states.

Table 1: Overview of the evolution of EU legislation on BSE surveillance by period (year and age of cattle*).

The implementation of a TSE surveillance program and transparent data reporting is a key component of the OIE geographical BSE risk assessment. The OIE provides a methodology to design TSE surveillance programs in cattle. The methodology considers the size and age distribution of the adult cattle population in countries and proposes one of two types of surveillance programmes according to an estimated prevalence of the disease to be detected.

• Type A surveillance was designed to allow the detection of BSE with an estimated prevalence of at least one case per 100,000 in the adult cattle population (confidence level of 95%).
• Type B surveillance was designed to allow the detection of BSE with an estimated prevalence of at least one case per 50,000 in the adult cattle population (confidence level of 95%).

Type B surveillance may be carried out in countries with negligible BSE risk status. It may also be carried out by countries with controlled BSE risk status following the achievement of the target prevalence using Type A surveillance.

While the OIE methodology remains the international standard for designing BSE surveillance programs its current validity is debated. Indeed, the model that was used to design the methodology relied largely on data derived from the c-BSE epizootic (i.e. the age of cattle detected as BSE-positive in the brain) observed in the 1980s and 1990s in the UK. These data no longer reflect the epidemiological situation of c-BSE as may exist in cattle populations.

Control

Following the emergence of the c-BSE epizootic in the UK, processed animal proteins produced from ruminant carcasses were rapidly identified as the main vector responsible for the spread of the disease in cattle populations. In this context, preventing the dietary exposure of cattle to ruminant tissues known to be susceptible to contain significant amounts of c-BSE infectivity appeared as an effective means to:

• prevent occurrence of new infection in cattle
• reduce the risk of c-BSE transmission to other species (farmed animals and human)

In order to achieve these goals, two specific and complementary types of measure were implemented, namely specified risk materials measures and the mammalian meat and bone meal (MBM) ban.

Specified Risk Materials measures

Specified risk materials (SRMs) refers to cattle tissues that are known to be susceptible to contain significant amounts of c-BSE prion infectivity. Elimination of the SRMs allows the exclusion of over 99.9% of c-BSE prion infectivity associated with infected cattle that are likely to enter the food chain. The OIE provides a list of SRMs that is considered to be the minimum standard of cattle tissue to be excluded. It is possible for countries to adopt more stringent regulations depending upon a number of national and international factors. The OIE has more flexible standards in terms of SRMs than those implemented in controlled BSE risk countries. Table 2 below provides an overview of the SRM in various geographical areas and guidelines from the World Organisation for Animal Health (OIE).

Table 2. EU 2015/728 amended 999/2001 from May 2015 which excluded the intestines from duodenum to rectum in cattle of all ages.

Apart from in the USA, SRMs are banned from both human food and animal feed chains. In the USA, while the full list of SRMs is banned from human food, only a subset of these tissues is banned from animal feed (i.e. the brain and spinal cord from cattle 30 months of age and older, as well as the entire carcass of BSE-positive cattle).

Currently available data suggest that SRM measures designed to deal with the c-BSE exposure risk are probably also efficient for mitigating the exposure risk to the L-BSE and H-BSE agents. However, the uncertainties that still exist about the distribution of infectivity in the tissues of atypical BSE cases preclude any quantitative estimate of the SRM measures.

Mammalian meat and bone meal (MBM) ban

In the UK a ban on the use of processed ruminant proteins to feed ruminants was introduced as early as 1988. In 1994 this feed ban was reinforced by the exclusion of all mammalian proteins (with specific exceptions) from ruminant feed material. In 1996 mammalian meat and bone meal (mammalian MBM) was banned from the feed for any farmed livestock (including fish and horses).

In 1994 the European Union introduced a ban of mammalian processed animal proteins (PAP) to cattle, sheep and goats. In January 2001 this feed ban was expanded to prohibit the use of all PAPs being fed to any farmed animal (with certain limited exceptions). This total feed ban was implemented to ensure that there was no cross-contamination between feed containing PAP intended for species other than ruminants and feed intended for ruminants. Only certain animal proteins considered to be safe, such as fishmeal, could be used for feed purposes (under very strict conditions). Table 3 below summarises the feed ban rules in the EU as applicable from 1 July 2017.

NA: Not authorized – A: Authorized under conditions

Table 3: Current measures related to the use of processed animal proteins in the European Union.

The feed ban measures that are in place in the EU exceed the OIE recommendations and those currently in place in most of the countries that experienced autochthonous c-BSE cases, such as the USA and Canada where convergent feed ban measures are now implemented.

The current OIE recommendations on feed ban includes:

• The removal of SRMs from the human food and the animal feed chains during slaughter and the processing of animal carcasses
• The prohibition of the inclusion of SRMs in animal feeds
• A ban on the use of PAP in ruminant feed (ruminant-to-ruminant feed ban, further reinforced by a mammalian-to-ruminant feed ban).

In 1997 Canada banned most mammalian proteins from ruminant feed. There were certain exceptions to this ban allowing porcine and equine meal to continue to be used in ruminant feed. In 2003 Canada enforced new measures that required the removal of the following cattle SRMs from the animal feed supply:

• Skull and brain, the trigeminal ganglia, eyes, tonsils, spinal cord and dorsal root ganglia of cattle over thirty months of age
• The distal ileum of cattle of all ages

Eradication

Historically when a c-BSE case was detected in the EU all the animals that belonged to the herd of origin of the index case were systematically destroyed. Numerous studies supported the contention that in the vast majority of the incidences, there were no other c-BSE cases present in the herd of origin, or in the same birth cohort as the index case. Current EU regulatory eradication measures consist of the culling, testing and elimination of:

• birth cohorts (bovine animals born within the 12 months preceding or following the date of birth of the affected bovine animal in the same herd)
• rearing cohorts (bovine animals that at any time during the first year of their lives were reared together with the affected bovine animal during the first year of its life)
• the progeny of a diseased female animal born within a period of 2 years prior to or after the clinical onset of the disease

11/03/2021 Updated by:

Olivier Andréoletti, ENVT, France