**2. Spread of BSE and vCJD**

Research into clinical BSE cases has reported that 84.3% of the BSE infectivity is associated with bovine CNS tissue.[14] Another study found that the CNS tissue carries 89.7% of the infectivity. [15] With regard to the pathogenesis of BSE, animal by-products such as meat and bone meal (MBM), which have been used as nutritional supplements in livestock feedstuffs for more than 100 years, are major carriers of the infectious agent PrPSc.[16, 17] Significant changes in the manufacturing processes of feedstuffs that took place in the 1970s, including the introduction of mechanical systems that permitted continuous flow production and solvent extraction of fats, enabled PrPSc to enter the livestock feed chain.[18, 19] The recycling of animal by-products from BSE-infected animals caused a wide spread infection of BSE in cattle,[19] subsequently affecting the food chain through the consumption of the PrPSc-contaminated meat products. [20] To date, more than 190,000 BSE cases have been reported in 28 countries since it was first identified in the UK in around 1986,[21] with approximately 97% of the cases being reported in the UK (Figure 1).[22] It has been estimated that the total number of BSE-infected cattle is around one million, of which around 75% entered the human food chain during the 1980s and early to mid-1990s.[23, 24] BSE has had a huge impact on the beef industry worldwide.[25-30] In the USA, estimates of the total loss of beef and offal exports ranged from \$3.2 billion to \$4.7 billion in 2004 after the nation's first BSE case was confirmed at the end of 2003.[31] Although the link between BSE and vCJD through eating prion-contaminated meat has not been conclusively proven, the consumption of food of bovine origin contaminated with PrPSc has been strongly linked to the occurrence of vCJD in humans.[20] Both vCJD and BSE share the same infectious agent, PrPSc.[32, 33] A total of 229 vCJD patients have been identified in 12 countries since it was first discovered in the UK in 1996 (Figure 1),[34, 35] of whom about 77% were in the UK (Figure 1).[36] It should be noted that following the successful containment of the BSE epidemic through the imposition of strict feed controls for ruminant animals, the numbers of cases of both BSE and vCJD in the UK have declined significantly since 1992 and 2000, respectively (Figure 1).

### **3. Contamination of SRM in meat products**

During the animal slaughter process, two major SRM, brain and spinal cord material, can easily contaminate meat products.[37-40] In general, there are four main pathways for the contami‐ nation of CNS-based SRM in meat products, which can occur at any stage during these processes: animal stunning, animal pithing, carcass dressing, and advanced meat recovery. The risks associated with each process have been studied and are discussed in turn below in

**Figure 1.** Number of reported BSE-cases (blue) and number of reported vCJD-cases (red) in the UK (1987-present)[22, 36]

the following sections. The major results of the relevant studies for each of these four pathways are summarized in Table 1.

#### **3.1. Animal stunning**

The stunning method has been widely used in many countries and areas of the world for some time as it slaughters the animals humanely. Stunning renders the livestock unconscious before slaughter, but because the heart of the stunned animal continues pumping for several minutes after stunning, during this time any CNS tissue that enters the jugular venous blood could still be spread throughout the body, contaminating both muscle and bone marrow via the blood circulation.[41] Several CNS markers that have a molecular weight similar to the PrPSc (MW: 30 kDa),[3] including syntaxin 1B and glial fibrillary acidic protein (GFAP),[42, 43] have been detected in non-CNS tissue after stunning. It thus seems likely that PrPSc could also be present in the edible carcass after stunning. Two types of captive bolt stunner, penetrative and nonpenetrative, are widely used to stun domestic animals prior to bleeding (Figure 2). Most abattoirs prefer to use penetrative captive bolt stunners, either with or without air injection, to render cattle quickly and painlessly unconscious before slaughter.



**Table 1.** Summary of the results of SRM contamination through the four major routes during animal slaughtering and meat processing

Table 1.

**3.1.**

**Animal stunning**

The stunning method has been widely used in many countries and areas of the world for some time as it slaughters the animals humanely. Stunning renders the livestock

unconscious before slaughter, but because the heart of the stunned animal continues pumping for several minutes after stunning, during this time any CNS tissue that enters the

jugular venous blood could still be spread throughout the body, contaminating both muscle and bone marrow via the blood circulation.[41] Several CNS markers that have a

molecular weight similar to the PrPSc (MW: 30 kDa),[3] including syntaxin 1B and glial fibrillary acidic protein (GFAP),[42, 43] have been detected in non-CNS tissue after

stunning. It thus seems likely that PrPSc could also be present in the edible carcass after stunning. Two types of captive bolt stunner, penetrative and non-penetrative, are widely

Summary of the results of SRM contamination through the four major routes during animal slaughtering and meat processing

unconscious before slaughter.

immunocytochemistry; <sup>e</sup>NF, neurofilament; <sup>f</sup>

NF-H), which are expressed mainly in neurons.[180]

sample size; <sup>i</sup>

tissues.[182]

3.1. Animal stunning

Table 1. Summary of the results of SRM contamination through the four major routes during animal slaughtering and meat processing

Note: NF is heteropolymer intermediate filaments that are composed of three neurofilament subunits (NF-L, NF-M and

S100 protein (MW: 10.5 kDa) is a calcium-binding protein found predominantly in the vertebrate nervous system.[181] Syntaxin 1B (MW: 33 kDa) which is an integral membrane protein exclusively and abundantly expressed in nervous

The stunning method has been widely used in many countries and areas of the world for some time as it slaughters the animals humanely. Stunning renders the livestock unconscious before slaughter, but because the heart of the stunned animal continues pumping for several minutes after stunning, during this time any CNS tissue that enters the jugular venous blood could still be spread throughout the body, contaminating both muscle and bone marrow via the blood circulation.[41] Several CNS markers that have a molecular weight similar to the PrPSc (MW: 30 kDa),[3] including

penetrative and non-penetrative, are widely used to stun domestic animals prior to bleeding (Figure 2). Most abattoirs prefer to use penetrative captive bolt stunners, either with or without air injection, to render cattle quickly and painlessly

IHC, immunohistochemistry; <sup>d</sup>ICH,

GFAP, glial fibrillary acidic protein; <sup>g</sup>NSE, neuron-specific enolase; <sup>h</sup>n,

<sup>a</sup>ELISA, enzyme-linked immunosorbent assay; <sup>b</sup>IB, immunoblotting; <sup>c</sup>

AMRS, advanced meat recovery system.

**Figure 2.** Classification of animal stunning techniques

Figure 2. Classification of animal stunning techniques

3.1.1. Penetrative stunning with air injection

#### This stunning technique involves the injection of compressed air into the cranium of cattle to effectively disrupt the brain *3.1.1. Penetrative stunning with air injection*

However, penetrative stunning with air injection poses a major risk of spreading the PrPSc from the BSE-infected CNS tissue to the edible carcass. Similarly, it is well known that severe penetrating injuries to the human brain, such as a gunshot wound, can cause acute brain damage and produce cerebral emboli that end up in the lung.[45] For each BSE-infected animal stunned with this type of stunner, there is a 31.2, 16.3, 3.3, and 0.7% probability that a portion of the PrPSc would be transferred to the blood, heart, lung, and liver, respectively,[44] which is significant This stunning technique involves the injection of compressed air into the cranium of cattle to effectively disrupt the brain structure. In 2001, it was estimated that 15% of cattle were stunned using an air-injection stunner in the USA.[44] However, penetrative stunning with air injection poses a major risk of spreading the PrPSc from the BSE-infected CNS tissue to the edible carcass. Similarly, it is well known that severe penetrating injuries to the human brain, such as a gunshot wound, can cause acute brain damage and produce cerebral emboli that end up in the lung.[45]

structure. In 2001, it was estimated that 15% of cattle were stunned using an air-injection stunner in the USA.[44]

For each BSE-infected animal stunned with this type of stunner, there is a 31.2, 16.3, 3.3, and 0.7% probability that a portion of the PrPSc would be transferred to the blood, heart, lung, and liver, respectively,[44] which is significant because it has been estimated that 60% of the liver, 50% of the heart, 25% of the kidney, and 5% of the blood are potentially available for human consumption.[44] Studies have also found that an air-injection stunner forced visible pieces of CNS tissue into the circulatory system of stunned cattle and sheep.[46, 47] This device has been shown to cause the formation of grossly visible brain tissue in the left and right branches of the main pulmonary artery in 2.5–5% of cattle,[46] in the jugular venous blood of cattle and sheep,[48-50] and in the aortic blood from sheep.[47] Particles of brain tissue as large as 20 μm in diameter can pass through the ovine pulmonary capillary network and enter the systemic arterial circulation during stunning, suggesting that this technique could easily disperse PrPSc throughout the edible carcass.[47] It has also been reported that CNS tissue has been observed in 33% of cattle hearts,[51] and in the kidneys of cattle stunned with air-injection stunners.[52]

Another factor to consider is that on occasion when operation of air-injection stunners fails, leading to an increase of CNS emboli formation and deposition, the probability of PrPSc transfer could be nearly 50% higher in blood, twice as high for the heart, and 10 times higher for the lung and liver.[44] Furthermore, a slaughterer who uses this type of stunner for too long or uses it successively to immobilize the same cattle could significantly increase the CNS tissue dispersion in blood vessels of the carcass.[51] It is also common for this type of stunner to press brain material out of the brain cavity through the bolt hole in the animal skull and splash onto the operator's face.[41, 53] Based on these results, the use of this type of stunner has been prohibited in many BSE-reporting countries and areas.

#### *3.1.2. Penetrative stunning without air injection*

This type of captive bolt stunner has a sharp-rimmed steel bolt and is powered by either compressed air (pneumatic-powered) or a blank cartridge that produces sufficient penetration force to initiate trauma to the cortex. It is the preferred tool for stunning cattle in the EU; 79 and 96% of the slaughterhouses in Europe use this method for cattle and calves, respectively. [54] However, the use of pneumatic-powered stunners and cartridge-fired stunners produced visible CNS clots in 12 and 1% of the cattle heart samples, respectively.[51] In addition, after using this type of stunner, CNS tissue was detected in the jugular venous blood from sheep, [49, 50] in venous blood from cattle,[55] in bovine pulmonary arterial system,[56] and on the surface of beef carcasses.[43]

In a study designed to trace the spread of CNS tissue in the cattle body after stunning with a cartridge-fired stunner, a marker microorganism, *Pseudomonas fluorescens*, was inoculated into the bovine brain via the stunning procedure.[57] The researchers reported finding this bacterium in the animal's blood, spleen, liver, kidney, lymph node, lung, and spinal cord. Using a similar approach, two microorganisms, *Escherichia coli* and *P. fluorescens*, have been detected in the blood, liver, lung, spleen, lymph node, longissimus muscle, and on the carcass surface of stunned sheep.[58] These studies demonstrate that the use of penetrative stunning without air injection could still spread CNS tissue throughout other portions of the carcass.

As with air-injection stunning, multiple use of this type of stunner also poses a high risk of spreading PrPSc from the infected brain to the edible carcass. In 2004, as few as 71% of slaugh‐ terhouses in the USA using penetrating captive bolt stunning successfully stunned 99–100% of the cattle with one shot and only 50% of abattoirs in Canada achieved the same target.[59] In 2005, this percentage decreased to 55% in US beef plants.[60] Since then, the percentage has increased gradually in both countries, reaching 91% in the USA by 2011.[61] In the EU, about 4–6.6% of captive bolt stunning in cattle requires a second stun.[54] Such practices could increase the risk of CNS tissue contamination in meat products. Although this type of stunner exerts less impulsive force on the stunned animal compared with air-injection stunners, it remains a threat to meat safety because this type of stunner is still in use worldwide.

#### *3.1.3. Non-penetrative stunning*

Non-penetrative stunning devices include the non-penetrating cartridge-operated stunner and the electrical stunner. Several studies have reported that no CNS tissue was detected in either aortic or venous blood samples collected from cattle or sheep after using these two devices.[48-50] However, other studies have detected brain tissue in venous blood samples from stunned cattle,[55] and on the surface of beef carcasses[43] after non-penetrating stunning. There also is an increased risk to operators as some animals may be inadequately stunned with non-penetrating stunning and recover their consciousness during slaughtering. Electrical stunning is the main alternative method for stunning cattle, but it is expensive to install and meat quality defects such as petechial hemorrhages have been linked to electrical stunning in sheep.[62]

#### **3.2. Animal pithing**

Pithing is the insertion of an elongated rod-shaped instrument into the cranial cavity of a stunned animal to further lacerate the CNS tissue.[63, 64] This operation prevents the animal stunned by a penetrating captive bolt from recovering permanently.[52] Pithing is considered by the industry to be a more effective technique for maintaining the safety of the operator and was used in 70% of UK abattoirs as recently as 1997.[50] However, in 2001, this technique, which has never been used in the USA,[52] was banned in the EU.[65] Brain tissue has been detected in venous blood[48, 50] and arterial blood samples[56] from cattle after pithing.

Based on scientific evidence of the dispersion of CNS-based SRM in response to various stunning and pithing techniques, the EU has ranked the stunning methods used in ruminants, from the most hazardous to the least dangerous, as: (1) penetrative stunning with air injection; (2) penetrative stunning without air injection; (3) captive bolt stunner with pithing; (4) captive bolt stunner without pithing and free bullet; (5) non-penetrative stunner and electro-narcosis. [66] Two techniques, air-injection stunning and pithing are now prohibited worldwide. However, there is still a 50% probability that emboli will be deposited in the blood of an animal stunned with a non-air-injection stunner.[44]

#### **3.3. Carcass dressing**

During animal slaughtering, the general sequence followed during the dressing procedure is stunning followed by exsanguination, dehiding, evisceration, splitting, washing, and chilling. [42] Of these steps, stunning is not the only point at which the carcass can potentially be contaminated with CNS tissue; other steps in the slaughtering procedure also have a high probability of dispersing CNS-based SRM across the carcass (Table 1). CNS tissue has been found in samples collected at a number of points along the slaughter line, including on the captive bolt gun, on the aprons worn by the operators, on their hands and the saws used, and in the longissimus muscle.[67]

In a study tracking two marker microorganisms, *E. coli* and *P. fluorescens*, both bacteria were transferred from the first stunned sheep to the stun wounds of the next 10 sheep stunned using the same contaminated penetrative cartridge-operated pistol.[58] These bacteria were also detected in the sheep's blood and on their carcass surfaces.[58] Perhaps most worryingly, the marker organisms were found in the air, and on the hands and aprons of the operator.[58] In another similar study,[57] the marker organism *P. fluorescens* was detected in the slaughter environment immediately after stunning and at each subsequent stage of the slaughterdressing process, including on the hands of the operators, the slaughter equipment (captive bolt gun and knife), the cattle hide, and the carcass splitting saw.

Spinal cord material is easily spread to bovine carcasses during carcass splitting. The majority of the CNS contamination was found on the internal surfaces of the carcass and along the cut vertebral surface, with lesser levels of contamination inside the body cavity.[68] CNS tissue was also detected in the surroundings during splitting, including on the hand held screen, the tray and apron, the captive bolt, the captive bolt aperture, and the floor of the receiving platform.[42, 69] Spinal cord tissue can also be transferred to subsequent bovine carcasses during carcass splitting. The main risk of subsequent carcass contamination comes from the splitting saw.[68] Overall, these studies demonstrate that the practice of carcass dressing, especially splitting, can lead to the extensive spread of SRM within the abattoir environment, contaminating equipment, surfaces, operators, and edible carcasses. There are also grounds for concern regarding operator safety when dealing with CNS tissue, particularly due to the aerosol of cerebrospinal fluid and spinal cord produced during the splitting process.

#### **3.4. Advanced meat recovery**

Mechanical systems have been developed to separate meat from bone by scraping, shaving, or pressing the meat from the bone, for example, via advanced meat recovery systems (AMRS). AMRS enables processors to remove any remaining muscle tissue from beef carcasses without breaking the bones.[70] About 70% of fed cattle and hogs, and 60% of cows slaughtered in the USA were processed using AMRS in 1998[71] and around 5,000 metric tons of mechanically recovered meat (MRM) were produced yearly during the period 1980–95 in the UK.[72] Although AMRS makes deboning more efficient and considerably faster than using a knife, if spinal cord material is attached to the spinal column as it enters these machines, it can be incorporated into the meat product produced.

A number of studies have detected CNS tissue in MRM products (Table 1).[73-76] In 2002, about 35% of US bovine MRM contained CNS tissue[77] and other studies detected spinal cord tissue in 29%[77] and 33.3%[75] of bovine MRM samples. Dorsal root ganglia tissue was found in 10% of the MRM samples.[77] Schmidt *et al.*[74] reported that 50% of the bovine MRM samples from 14 slaughter plants that they tested were positive for CNS protein. In a later study by the same group,[76] 17.2% of the MRM samples tested were contaminated by CNS tissue.

By the 1990s, about 90% of the beef MRM produced was being used in burgers in the UK.[78] Around 75–80% of individuals surveyed in France consumed MRM products from burgers, and the consumption of burgers increased by 40% over the period 1980–1995.[79] Frequent consumption of beef and beef products, including burgers and meat pies, containing the MRM or head meat has been linked to an increased risk for vCJD;[80] infection with vCJD is thought to be predominantly due to exposure to BSE in beef MRM and head meat products.[81]

Looking at the results of the present review (Table 1), every stunning method except for electrical stunning could potentially contaminate meat products by dispersing CNS-based SRM, including any PrPSc present in sub-clinically infected cattle. Although the use of airinjection stunning, pithing, and AMRS has now been prohibited during animal slaughtering and meat processing (Table 2), penetrative stunning without air-injection and non-penetrative stunning are still in widespread legal use. Slaughter conditions and procedures such as carcass dressing, especially the splitting process, can also result in widespread contamination within abattoirs, contaminating equipment, surfaces, operators, and carcasses destined for human consumption. CNS contamination of bovine carcasses simply cannot be eliminated by the slaughtering technology currently used.
