Bovine Spongiform Encephalopathy (“Mad Cow” Disease)

Christine F. Berthelin-Baker • John R. Middleton

■ Definition and Etiology Bovine spongiform encepha­lopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle that was first recognized in 1986 in Great Britain.¹ The TSEs are a group of slowly progressing, invariably fatal, neurodegenerative diseases that can affect humans and animals.

■ Epidemiology Current evidence indicates that BSE resulted from a single agent.^5-10 BSE may have originated from feedstuffs containing rendered carcasses of scrapie-infected sheep, or it might have been a preexisting sporadic and rare disease unrecognized in the bovine population. The BSE epi­demic was traced to the recycling of BSE-infected carcasses into cattle feed, which occurred mostly in the 1980s in the United Kingdom.⁶ Cattle in other countries were affected as a result of the importation of infected feedstuffs or cattle, followed by later recycling the agent in their own rendered ruminant feedstuffs. At the time of writing, the OIE reported that BSE had been detected in 28 countries, including the United States.¹¹ Since 1986, more than 181,000 cases of BSE have been confirmed in Great Britain.¹¹ These cases were distributed among 37,170 farms.¹² The incidence of BSE was highest among the cattle cohorts born between 1986 and 1989, and the outbreak peaked in 1992, when 36,680 cases were detected by screening suspected clinical cases (passive surveillance) in Great Britain.

BSE has affected predominantly dairy cattle because of their longer life span and feeding practices, which in Great Britain included feeding animal-derived protein to youngstock. No clear genetic predisposition to BSE has been demonstrated in cattle, in contrast to scrapie in sheep, although offspring of affected cattle seem to be at increased risk for the disease. The epidemic (classical) form of the disease is considered to be a foodborne disease of cattle, associated with the ingestion of meat and bone meal contaminated with the BSE prion. This is substantiated by the steady decline in the incidence rate of BSE in endemic countries since the enactment of bans on feeding ruminant protein to ruminants.¹¹

Rigorous, active surveillance programs have been initiated in many countries to screen cattle and beef products for the BSE agent. As a result of this large-scale testing, two extremely rare prions were discovered on the basis of protease resistance patterns and glycopatterns.^13-16 These novel agents have been termed H-BSE and L-BSE, and animals from which these agents have been identified are said to have atypical BSE.⁵ These cases have been detected in multiple countries by rapid prion detec­tion tests conducted on aged (>8 years) cattle that were tested either during slaughter surveillance or through targeted surveil­lance of downer cattle.

■ Pathogenesis The exact function of the normal prion protein is unknown, as are the mechanisms of abnormal prion protein (e.g., PrP-BSE) induced neurodegeneration and CNS dysfunction in TSEs. On the basis of research in transgenic mice expressing bovine PrP, the PrP-BSE is thought to undergo limited propagation in the distal ileum before spreading to the CNS, probably by moving through the gut-associated lymphoid tissue into the peripheral nerves.^17,18 Studies of naturally infected cattle, combined with experimental oral challenge studies in cattle, have revealed that BSE infectivity may exist in the brain; spinal cord; retinal tissue; optic nerve; cervical, thoracic, and trigeminal ganglia; facial and sciatic nerves; and the distal ileum. The skeletal muscles, spleen, and lymphatic tissues did not demonstrate infectivity.^19,20 Intrace­rebral inoculation of cattle has also been demonstrated to result in infectivity of the tonsillar tissue.²¹ After a prolonged incuba­tion period, accumulation of PrP-BSE in the CNS creates progressive and irreversible neurologic dysfunction.

■ Clinical Signs As with all TSEs, BSE has a long incuba­tion phase. It occurs mostly in 4- to 6-year-old cattle (age range, 20 months to 18 years), although the atypical forms tend to occur in cattle older than 8 years. The clinical signs of BSE usually have an insidious onset.^22,23

The detection of clinical signs of BSE and the array of clinical signs reported are influenced by how closely the cattle are being observed and the handler's awareness of the insidious and nonspecific nature of the clinical signs. Subtle early behavioral changes are unlikely to be observed in large herds. Affected cattle may become apprehensive, stay apart from their herdmates, and become fearful of their handlers.

Other possible clinical signs of BSE include ptyalism, increased head rubbing, muscle fasciculations, and excessive vocalization. Other behaviors observed in animals with BSE may include frequent and repetitive head tossing, licking of the nostrils, yawning, flehmen response, head butting, and restlessness.²⁴ Because of the hyperexcited state of these animals, tachycardia is expected; however, relative bradycardia (60 beats/ min) may be observed. Affected animals often lose weight and have reduced milk output. Ataxia and tremors occur in more advanced cases. Tremors most often involve the head and neck and may become generalized, especially with exercise. Ataxia is most marked on turns, when the animal goes over steps, or on uneven terrain; it may also be accentuated with exercise. Animals with BSE may become recumbent and unable to feed if the disease is left to run its course.

■ Diagnosis No in vivo test is approved for the diagnosis of BSE. The diagnosis can be confirmed only by postmortem examination and is based on microscopic brain examination or tests that identify the abnormal prion in brain or spinal cord tissue.^1,27 Rapid prion tests include Western blot, paraffin- embedded tissue (PET) blot, and ELISA.^28-31 Rapid tests have enabled the detection of BSE through surveillance in targeted at-risk populations such as fallen (downer) cattle.

Histopathologically, BSE is characterized by neuronal degeneration and intraneuronal vacuolation in specific brain areas. The vacuolation is accompanied by or preceded by the accumulation of PrP-BSE.^6,7,32 The obex of the medulla is currently considered to be the tissue of choice for detection 33

of the BSE agent.³³

Nervous system diseases that could be confused with BSE include viral encephalopathies (pseudorabies, rabies, Borna encephalitis, bovine immunodeficiency virus encephalitis), listeriosis, polioencephalomalacia, lead poisoning, CNS parasitic migration, brain tumors and abscesses, and vitamin A deficiency. Hepatic encephalopathy and other metabolic imbalances may also be confused with BSE. Because of the insidious nature of BSE, affected cattle may appear to recover coincidentally with blanket therapy for suspected metabolic imbalances and later exhibit relapse. This must be kept in mind whenever therapy for metabolic imbalances initially appears to be successful but is followed by one or more relapses.

■ Treatment and Control BSE is always fatal, and no treatment is available.

Various measures have been taken to protect public health by removing potentially infected animals and tissues from the food chain. The disposal of BSE-infected tissues and the sourcing of bovine tissues and body fluids for the preparation of medicinal products must take into account the extreme resistance of TSE agents. All TSE agents remain infectious after exposure to a wide range of inactivating treatments and environmental changes such as autoclaving, rendering, storage for months to years, exposure to ultraviolet light, freezing, thawing, prolonged boiling, and incubation with formalin.^34-36 Because of the public health risk, the carcasses of animals suspected of having BSE should be disposed of in an alkaline- hydrolysis tissue digester designed to inactivate TSE agents.³⁷ In countries that have had an outbreak of BSE, the complete exclusion of ruminant tissues from ruminant feed has aided progress toward eradication of BSE, as previously noted. In the United Kingdom the initial feed ban of 1988 was found to be insufficient and was reinforced by successive measures. The 1996 ban on feeding mammalian meat and bone meal to all farmed animals was introduced to prevent contamination of ruminant feed at feed mills and down the supply chain to the farm. Because of the long incubation period of BSE, the implementation of feed control measures did not reduce the incidence of this disease immediately.

■ Public Health Consideration and BSE in Other Nonbovine Hosts Since the onset of BSE epidemics, new TSEs have been identified in animals other than cattle. Strain typing in mice showed that these new TSEs were caused by the BSE agent,^38,39 which has proved to be a relatively promiscu­ous TSE agent, capable of experimentally infecting mice, sheep, goats, pigs, cats, mink, and primates.⁵ The disease in humans, termed new-variant Creutzfeldt-Jakob disease (vCJD), was first identified in 1996 in the United Kingdom, where it had affected 178 people as of April 2018.⁴⁰ Other countries have detected vCJD cases in people who had lived in the United Kingdom between 1980 and 1996, as well as in citizens who never traveled to the United Kingdom. The emergence of vCJD has put worldwide emphasis on the prevention and detection of TSEs in animals. The route of infection of people by BSE has not been clearly traced, but a case-control study demonstrated that the consumption of food that included infected cattle nervous tissue was associated with increased risk of vCJD.⁴¹ This and other evidence has led to successive “Specified Risk Materials” bans in many countries, aimed at preventing contamination of human food with nervous system or other specified high-risk tissues from ruminant animals.

Scrapie

■ Definition and Etiology Scrapie is a TSE that naturally affects sheep, goats, and mouflons. It is the oldest known TSE, described more than two centuries ago. The causative agent is considered to be an abnormal prion (PrP-Sc) distinct from that of BSE.¹ At least three distinct types of the scrapie agent have been identified in the natural hosts.¹ Although sheep are susceptible to experimental challenge with the BSE agent, natural BSE has not been reported in sheep.¹

■ Epidemiology Scrapie occurs endemically in sheep flocks worldwide, with the exception of Australia and New Zealand.¹ Sheep are the natural host of scrapie, but the infection can be maintained in goats that have no direct contact with sheep, which indicates that transmission in goats is both lateral and vertical.^2-4 Scrapie cases occur sporadically in infected flocks. Only one or a few animals are affected at any given time. Outbreaks may occur if scrapie is introduced into a genetically susceptible flock.

Although no breed is known to have a predisposition to scrapie, the genetic makeup of the host controls differences in susceptibility and resistance to scrapie in various breeds.^1,5,6 The molecular basis of this resistance is largely controlled by the PrP gene.⁷ Polymorphisms at three codons of this gene (136, 154, and 171) appear to be the main determinants of the susceptibility of sheep to scrapie.¹ According to current evidence, the genotype VV₁₃₆RR₁₅₄QQ₁₇₁ (or AA₁₃₆RR₁₅₄QQ₁₇₁ in some breeds) is most susceptible, and the genotype AA₁₃₆RR₁₅₄RR₁₇₁ is highly, but not completely, resistant.¹ Susceptibility is most clearly linked with variation at codons 136 and 171 in breeds studied to date; the genotype of a particular animal is often referred to as RR, QR, or QQ to designate the alleles at site 171. Pedigree, phenotypic expression of the disease, or genetic testing may be used to identify sheep with “resistant” PrP genotypes. Genetic polymorphisms or the PrP gene may also influence susceptibility to scrapie, the incubation period for scrapie, or both in goats.⁸

■ Pathogenesis In contrast to BSE, which is restricted to the gut-associated lymphoid tissue and nervous tissue, the scrapie agent can be identified in infected sheep in various nonneural tissues such as those of the lymphoreticular system, kidney, and placenta.^9,10 This difference has facilitated the development of lymphoid tissue biopsy techniques to detect scrapie (described later). Most transmission of scrapie appears to take place during the birthing season.¹ The scrapie agent is probably spread by the placentas and birthing fluids of infected animals,^11,12 and oral uptake is considered the probable route of entry.^11-13 Transport of PrP-Sc to the CNS probably occurs along the vagus nerve from the GI tract,¹⁴ and in most cases the agent can be demonstrated in the vagal nucleus of the brainstem early in the stage of neuroinvasion.¹⁵ It then spreads into higher brain centers. Dysfunction of the CNS appears to correlate approximately with accumulation of PrP-Sc in the brain; however, subtle dysfunction can occur with little or no characteristic histologic changes. The exact mechanism that causes neurologic dysfunction remains to be fully defined.⁹

■ Clinical Signs Most animals with scrapie are 1 to 5 years old. The clinical course varies but is typically slow and may last several months. Early behavioral changes are often accom­panied by weight loss. Animals with scrapie may stay apart from the flock and become nervous and restless.¹⁶ Pruritus of increasing intensity is a common feature; affected sheep may rub on fixed objects, scratch with their horns or hooves, and bite or lick themselves excessively. The head, withers, flanks, back, rump, base of the tail, and lower limbs are typically affected, showing secondary wool loss, dermatitis, and skin infections or excoriations.^2,16 Head and face rubbing and shaking may also cause ocular lesions or aural hematomas. When scratching the pruritic areas (mostly the back and rump), the animal may display a “scratch reflex” (or “nibble reflex”), which may include reflex nibbling, lip licking, and rhythmic head movements. This reflex is often, but not always, present in scrapie cases. This response may also be observed in other CNS diseases and in pruritic skin diseases, particularly ecto­parasitism, so it should not be considered specific for scrapie.

Other clinical signs of scrapie include bruxism, ptyalism, and regurgitation. Tremors may start with the head and general­ize to the whole body. Apathy, exercise intolerance, hypermetria, and ataxia may develop. Animals with the most severe and advanced cases show stupor, episodic collapse, and convulsions. Death may occur during a convulsion or as a result of starvation. The clinical signs of scrapie in goats are broadly similar to those in sheep.^2,3,17-19 Scrapie has been identified in fallen (down) sheep as well.

■ Diagnosis Because the scrapie agent can disseminate in lymphoid tissue before the development of clinical disease, immunohistochemistry studies may show PrP-Sc in biopsy samples containing lymphoid tissue from the tonsils,²⁰ nictitating membranes,²¹ retropharyngeal lymph nodes,²² or rectoanal junction.^23,24 However, these tests have limited sensitivity, primarily because of variable lymphoid involvement among infected sheep, a phenomenon that may be associated with the host's genetic makeup.²⁵ Microscopic examination of the brain and spinal cord is the classic diagnostic method for scrapie. The scrapie agent is possible to identify only when tissues are collected soon after death, before autolytic changes take place. Neuronal vacuolation and PrP-Sc deposits are found in specific brain nuclei.^26-31 Collection of brainstem tissue (particularly the obex), tonsillar tissue, and medial retropharyngeal lymph nodes is currently recommended for testing suspect sheep in the United States.³² In autolyzed tissues, PrP-Sc can be identified by immunohistochemistry testing, Western blot tests, or ELISA.^33-36 As with BSE, heightened active surveillance for scrapie has revealed at least one atypical strain of scrapie (termed atypical scrapie).³

Nervous system diseases that could be confused with scrapie include pseudorabies, rabies, Borna encephalitis, listeriosis, polioencephalomalacia, lead poisoning, cerebrospinal nemato- diasis, gid (coenurosis), brain tumors, brain abscess, maedi-visna virus infection, vitamin A deficiency, pregnancy toxemia, and other metabolic imbalances. When pruritus is the only clini­cal sign, skin diseases such as psoroptic and sarcoptic mange, ringworm, myiasis, pediculosis, and atopy may be considered in the differential diagnosis. Because of the insidious nature of the illness, animals with early scrapie may initially appear to respond to a variety of treatments. This must be kept in mind whenever specific therapy for another suspected illness appears initially to be successful but is followed by relapse. When therapy of an alternative condition fails, the final diagnosis must rely on detailed examination of postmortem samples, as discussed previously.

■ Treatment and Control Scrapie is a fatal and irreversible disease for which there is no known treatment.

Because there is no effective treatment for scrapie, control measures designed to prevent the spread of the disease are especially important. Scrapie is a reportable disease. Eradication measures vary from country to country. Animals suspected of having scrapie are destroyed. Contaminated pastures or pad­docks may be left empty of livestock, but the scrapie agent is unlikely to be inactivated in the environment. Contaminated stalls, corrals, and sheds should be disinfected with sodium hypochlorite diluted at 4% available chlorine. In the United States and Canada, scrapie-affected and related animals are destroyed, and their flocks are quarantined. Lines of sheep genetically resistant to the development of clinical scrapie have been developed,^38,39 and genotype testing is currently used to select for resistance to the disease.¹

■ Public Health Considerations Although scrapie has been endemic in various areas of the world for more than two centuries, epidemiologic studies have not shown any correlation between the incidence of Creutzfeldt-Jakob disease in humans and that of classical scrapie in sheep; the zoonotic potential of atypical scrapie remains under investigation.¹ It is nevertheless prudent to take precautions to minimize the potential for human exposure to scrapie because of the high fatality rate characteristic of TSEs and the potential for emergence of novel strains of prions with unknown host ranges.¹