Brain tissue fragments in jugular vein blood of cattle stunned by use of penetrating or nonpenetrating captive bolt guns

Although the incidence of bovine spongiform encephalopathy in cattle continues to decline in the United Kingdom, it remains important to maintain vigilance of all potential routes of transmission of infection to humans. Initial studies have demonstrated a potential risk of carcass contamination with brain tissue following the use of captive bolt gun stunning in cattle. The objective of this study was to further explore these initial findings particularly in regard to captive bolt guns currently in use in the United Kingdom. Brain tissue fragments or elevated levels of a marker protein for brain tissue were detected in venous blood samples from 4% (95% confidence interval, 1.6 to 9.8%) of cattle stunned by penetrating captive bolt gun and from 2% (95% confidence interval, 0.6 to 7%) of those stunned by nonpenetrating captive bolt gun.     

Recent concerns about stunning and slaughter

This review summarises information that is relevant to concerns that have recently been expressed about stunning and slaughter. It is known that captive bolt stunning can result in brain material passing to the lungs via the jugular veins. If future studies show that BSE prions pass beyond the lungs to the edible carcass, there will be a move away from captive bolt stunning in large cattle towards electrical stunning. Greater use of electrical stunning in large cattle will increase the importance of blood splash in the beef industry. The theoretical causes of blood splash are reviewed to improve our understanding of this problem. In some situations it can be due to excessive venous pressure causing rupture of a capillary bed some distance from the source of the pressure rise, but it is not known whether this applies to electrical stunning. Gas stunning is replacing electrical stunning for poultry because it can reduce blood spots, which is a similar condition to blood splash. Several gas stunning methods are now being used, but it is not clear which of these is the most humane. Anoxic stunning leads to carcass convulsions and this causes more carcass damage. In fish, recent developments in electrical stunning are showing promise in overcoming problems with carcass damage. It is recommended that rock lobsters should be chilled or frozen before butchery, to ensure a humane death.     

Dissemination of central nervous system tissue from the brain and spinal cord of cattle after captive bolt stunning and carcass splitting

In the absence of reliable live animal tests for the presence of BSE in cattle, a number of measures have been applied to exclude specified risk materials (SRM) from the human food chain. However, concerns remain that current practices in the stunning and slaughter of cattle may disseminate central nervous system (CNS) tissue to meat and meat contact surfaces. The objective of this study was to establish the particular risks of CNS tissue dissemination associated with captive bolt stunning and carcass splitting. The study applied enzyme linked immunosorbent assays (ELISAs) in the detection and quantification of two CNS proteins, syntaxin 1b and GFAP. The study observed extensive dispersal of both CNS proteins onto equipment, beef hide and personnel. These results demonstrate that despite the rigorous application of current SRM control policies, normal slaughter practices continue to present significant opportunities for CNS material including BSE prion present in the CNS of any sub-clinically infected cattle to contaminate meat entering the human food chain.     

Comparison of commercially available antibodies for the detection of central nervous system tissue in meat products by enzyme-linked immunosorbent assay

An indirect competitive enzyme-linked immunosorbent assay was used to compare four commercially available monoclonal and polyclonal antibodies prepared against the markers of central nervous system tissue: glial fibrillary acidic protein, neurofilaments, and myelin basic protein. These antibodies were investigated for their usefulness at the detection of prohibited central nervous system tissue in meat products to prevent spread of bovine spongiform encephalopathy and its human form, a new variant of Creutzfeldt-Jakob disease. The detection limit was 2.5 to 10%. The specificity was tested using extracts of tissue samples of bovine and porcine brain, spinal cord, lymphatic node, diaphragm, skeletal muscle, and peripheral nerves. All antibodies reacted with both bovine and porcine tissue extracts.     

Glial fibrillary acidic protein and myelin basic protein as markers for the immunochemical detection of bovine central nervous tissue in heat-treated meat products

Because bovine central nervous tissue (CNT) is the main risk material in transmission of the infective agent of bovine spongiform encephalopathy, a suitable test is needed to enforce the ban on CNT in human foodstuffs in the United States and the European Union and to ensure that meat products are free of CNT To detect bovine CNT in heat-treated meat products, we used immunohistochemistry and Western blots with antibodies against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). Both antigens were resistant to processing methods used for meat products. The anti-GFAP antibody showed a high degree of tissue specificity, whereas the anti-MBP antibody had high species specificity, clearly differentiating between porcine and bovine CNT Therefore, immunochemistry performed with both proteins provides an effective means for detecting bovine CNT in meat products.     

Comparison of analytical methods for the detection of central nervous system tissue in ground beef

Bovine spongiform encephalopathy is most likely transmitted by the consumption of central nervous system tissue of infected cows. The objective of this study was to compare the sensitivity or limits of detection of two central nervous system tissue detection assays (glial fibrillary acidic protein enzyme-linked immunosorbent assay [ELISA] and neuron-specific enolase Western blotting assay) in by-products and ground beef. By-products including brain, spinal cord, and ileum were collected from the slaughterhouse and used for analyses with ELISA and Western blotting assays. Beef samples were prepared by mixing ground beef with different amounts of spinal cord tissue (0, 0.03, 0.06, and 0.1%) and were analyzed using the two central nervous system tissue detection methods. Both analytical assays were applicable in detecting central nervous system tissue in ground beef. However, the ELISA method was considered superior because of its ease of use, high sensitivity, and rapidity as compared with the Western blot method.     

Comparison of stunning methods in the dissemination of central nervous system tissue on the beef carcass surface

Beef carcasses were examined to explore the effects of stunning methods on central nervous system tissue (CNST) dissemination on the surface during the slaughtering process. The frequency of occurrence of CNST contamination on four defined parts, each on the interior and exterior surfaces of the split carcass, and their level of the contamination were measured by an ELISA test. The effect of hot carcass weight was also examined. The results showed that the frequency of contamination occurrence was not affected by the stunning method. However, the penetrating captive bolt stunning method resulted in a higher level of CNST contamination than non-penetrating sledge-hammer stunning (P < 0.001). The higher level of contamination occurred on the interior surface of carcasses along the vertebral area. Therefore, the dissemination of CNST on carcasses seemed to be affected by the stunning method, carcass splitting, and carcass washing. The carcass weight significantly affected the level of CNST contamination on the carcass (P < 0.01) but there was no interaction with the stunning method.     

Transfer of spinal cord material to subsequent bovine carcasses at splitting

During the slaughter process, cattle carcasses are split by sawing centrally down the vertebral column, resulting in contamination of each half with spinal cord material. Using a novel method based on a real-time PCR assay, we measured saw-mediated tissue transfer among carcasses. Up to 2.5% of the tissue recovered from each of the five subsequent carcasses by swabbing the split vertebral face came from the first carcass to be split; approximately 9 mg was spinal cord tissue. Under controlled conditions in an experimental abattoir, between 23 and 135 g of tissue accumulated in the saw after splitting five to eight carcasses. Of the total tissue recovered, between 10 and 15% originated from the first carcass, and between 7 and 61 mg was spinal cord tissue from the first carcass. At commercial plants in the United Kingdom, between 6 and 101 g of tissue was recovered from the saw, depending on the particular saw-washing procedure and number of carcasses processed. Therefore, if a carcass infected with bovine spongiform encephalopathy were to enter the slaughter line, the main risk of subsequent carcass contamination would come from the tissue debris that accumulates in the splitting saw. This work highlights the importance of effective saw cleaning and indicates that design modifications are required to minimize the accumulation of spinal cord tissue debris and, hence, the risk of cross-contamination of carcasses.     

A comparison of the effects of electrical, captive bolt or percussion stunning on the incidence of blood splash in lambs

Three-hundred-and-three lambs were divided into three equal groups. Half the lambs in each group were not weaned before slaughter and the weaned half were slaughtered a week later. Lambs in one group were head stunned electrically before slaughter; in the second group lambs were stunned with a captive bolt pistol and in the third group they were percussion stunned before slaughter. Carcasses, gall-bladders, hearts and duodenums were inspected for blood splash after slaughter. A few small haemorrhages were detected in 57% of the electrically stunned lambs, in 30% of the captive bolt stunned lambs and in only 16% of the percussion stunned animals. Although no splash of commercial significance was found in any carcass, 25% of the electrically stunned lambs had detectably splashed carcasses, whilst only 1% of percussion-stunned lambs had splashed carcasses with captive bolt stunned lambs being intermediate in carcass splash incidence. These results show that stunning method affects the incidence of blood splash.     

Evaluation of an enzyme immunoassay for the detection of central nervous system tissue contamination at the slaughterhouse

To protect public health from bovine spongiform encephalopathy, European Commission Regulation EC 1139/2003 on monitoring programs and specified risk material requires that as of 1 October 2003, each member state has in place a sampling plan with an appropriate laboratory test to detect central nervous system (CNS) tissue in bovine head meat harvested at slaughterhouses or cutting plants. With this study, we wanted to evaluate the accuracy and reliability of an enzyme immunoassay, the RIDASCREEN Risk Material 10/5, in targeting a CNS-specific marker, the glial fibrillary acidic protein. A receiver operating characteristics curve was plotted to identify the best cutoff of CNS concentration. Reliability was calculated by Cohen's kappa on data from two diagnostic sessions. Test performance showed high sensitivity and specificity (97.9 and 97.4%, respectively) for a cutoff value between positive and negative at a CNS concentration of 0.049%; reliability of test precision was also very good. When these criteria are applied, the RIDASCREEN Risk Material 10/5 test appears to be a reliable tool for monitoring CNS tissue contamination in meat. This diagnostic procedure should therefore be recommended for national application in monitoring programs.     

Animal welfare at markets and during transport and slaughter

This review highlights some recent developments in our understanding of stress and physical injuries that occur before and during transport to slaughter, during handling at livestock markets, and at the time animals are put-up for slaughter within abattoirs. Stress in pigs during transfer to the stunning point within the abattoir has important effects on meat quality, and there is growing evidence that strenuous exercise or CO(2) stunning can contribute to oxidative rancidity in pigs, poultry and fish. In the EU, putting cattle through a crush in order to check that their eartag numbers correspond to their passport numbers is imposing additional stress, and there are reports that it is leading to greater hide contamination with Escherichia coli O157. Recent developments in stunning and slaughter include a better understanding of the causes of variation in captive bolt gun performance, the effectiveness of poll instead of frontal shooting in water buffalo, the prevalence of false aneurysms in carotid arteries during shechita and halal slaughter, and the stress effects of CO(2) stunning in fish. Stunning pigs with 90% CO(2) leads to less PSE meat than 80% CO(2). There have been concerns about the physical activity that cattle show following electrical stunning with an electrically induced cardiac arrest, and with electrical stunning using DC waveforms in broiler chickens. There is also growing concern about the hygiene problems that exist in wet markets, where animals are slaughtered alongside meat that is on display to customers.     

The influence of slaughter method on the residual blood content of meat

The effect of slaughter method on the residual haemoglobin content of the M. longissimus dorsi and the amount of blood lost at exsanguination of sheep was investigated. The influence of stunning method (electrical, 90 V, or captive bolt pistol), pithing and bleeding-out position were examined. Sheep bled lying lost more blood than those bled hanging and electrically-stunned animals lost more blood than those stunned with a captive bolt pistol; pithing had no effect. The residual haemoglobin content of the muscle did not vary significantly between methods and there was no relationship between it and the amount of blood lost at slaughter.     

Identification of central nervous system tissue in retail meat products

A procedure to detect tissues from the central nervous system that involved quantification of cholesterol and immunochemical detection of neuron-specific enolase and glial fibrillary acidic protein was used to analyze 402 samples of heat-treated meat products from various food outlets in Germany. The cholesterol content of 16 samples (4.0%) indicated the possible presence of central nervous system tissue because the levels exceeded the normal maximum cholesterol content of cooked sausages. In 7 of these 16 heat-treated meat products, immunoblotting of both neuron-specific enolase and glial fibrillary acidic protein confirmed the presence of CNS tissue. Repeated sampling by veterinary officials and analysis by both cholesterol quantification and immunoblotting confirmed these findings. Whereas all of the control samples (with and without added central nervous system tissue) were correctly classified by both cholesterol quantification and immunoblotting, negative results of immunoblotting must be carefully interpreted in the case of intensively heat-treated meat products. Thus, studies have yet to establish an increase in sensitivity of immunoblotting of neuron-specific enolase and glial fibrillary acidic protein. However, the detection of illegal use of central nervous system tissue in heat-treated retail meat products demonstrates the need for suitable analytical methods to control transmissible encephalopathies and to enforce labeling laws.     

Post-mortem variation in pH, temperature, and colour profiles of veal carcasses in relation to breed, blood haemoglobin content, and carcass characteristics

A total of 1764 male calves of the Meuse-Rhine-Yssel (MRY) and Friesian-Holstein (FH) breeds were slaughtered at two commercial slaughterhouses to investigate the variation in pH, temperature, and colour of Dutch veal carcasses processed without electrical stimulation and with a moderate chilling regimen (average temperature of the longissimus lumborum muscle at 45 min, 3, 24, and 48 h post-mortem was 38.4, 23.3, 3.7 and 1.9°C, respectively). Blood haemoglobin content was determined 2 weeks before slaughter. The efficacy of captive bolt stunning was scored and the carcass movements after shackling registered. Temperature and pH measurements were carried out at 45 min, and 3, 24, and 48 h after slaughter in the longissimus lumborum muscle (LL). Muscle surface colour (CieLAB-values) was measured with a colorimeter at the rectus abdominis muscle at the same times post-mortem. Carcass conformation and visual carcass colour classification were determined at 45 min post-mortem. Carcasses with a better EUROP-conformation score and heavier weight showed a higher rate of pH decline and a slower cooling rate. Slight differences in cooling systems between the slaughterhouses caused marked differences in pH and temperature profiles. Significant differences in carcass weight were observed between the MRY and FH breeds, leading to significant differences in pH and temperature profiles. Carcass colour of the two breeds was similar. Veal carcass colour in general was not related to the observed variation in post-mortem pH and temperature in the longissimus lumborum muscle and was shown to be more associated with the blood haemoglobin content. Haemoglobin content of the blood in the period before slaughter was shown to be related to the visually assessed carcass colour at 45 min post-mortem (using a 10-colour scale), as well as to the instrumentally determined L*-value, with significant correlation coefficients of 0.61 and -0.61, respectively. Repeated captive bolt stunning to obtain unconsciousness significantly decreased pH at 3 h post-mortem. The degree of carcass movement after slaughter did not influence pH, temperature, or colour profiles of the carcasses.     

Blood in the respiratory tract during slaughter with and without stunning in cattle

Bovine respiratory tracts were examined for blood following shechita without stunning, halal slaughter without stunning, and captive bolt stunning with sticking. In all three methods the cattle were in the upright (standing) position at the start of bleeding. Those that had not been stunned continued to breathe during the early part of bleeding whilst those that were stunned were not breathing. Nineteen percent of the shechita, 58% of the halal and 21% of the stunned plus stuck cattle had blood lining the inner aspect of the trachea. Thirty six percent, 69% and 31% had blood in the upper bronchi, respectively. Ten percent, 19% and 0% had fine bright red blood-tinged foam in the trachea, respectively. It was concluded that concerns about suffering from airway irritation by blood could apply in animals that are either not stunned before slaughter or do not lose consciousness rapidly whilst blood is present in the respiratory tract.     

The effects of electrical stunning and percussive captive bolt stunning on meat quality of cattle processed by Turkish slaughter procedures

The effects of electrical and percussive captive bolt stunning both on initial and up to 14 days post mortem meat quality of Friesian young bulls were studied. The first group (NS; n=10) were slaughtered without stunning under Turkish slaughter procedure (these animals were slaughtered under conditions acceptable to the appropriate ethics committee) in a modern abattoir. In the second group (ES; n=10) animals were electrically stunned. In the third group (PS; n=10) animals were stunned with percussive captive bolt. Meat quality was assessed by examining muscle glycogen concentration, pH and WHC. Also cooking loss, texture parameters and consumer sensory properties were assessed at 24 h, 4, 7 and 14 days post mortem. Meat colour as L^*, a^*, b^*, C^*, h^* values were evaluated at 0, 48 h, 3, 5, 7, 9 and 14 days post mortem. Pre-slaughter handling affected muscle glycogen concentration (P<0.05). Animals in electrical and percussive captive bolt stunned groups had higher muscle glycogen concentrations compared to animals in the non-stunned group (P<0.05). Although pre-slaughter handling did not affect significantly pH_{24 h} and WHC, differences were significant for cooking loss, colour coordinates and texture parameters at some storage times. For all sensory attributes (odour, flavour, tenderness and overall acceptability) at all ageing periods (24 h, 4, 7 and 14 days) the percussive stunned (PS) group was significantly superior to the non-stunned (NS) group. The electrically-stunned (ES) group also tended to be superior to the NS group although not all differences were statistically significant. These results indicate that percussive captive bolt stunning of cattle improved meat quality compared with cattle electrically stunned using head only tongs and those non stunned under approved Turkish slaughter procedure.     

A comparison of the risk of bovine spongiform encephalopathy infectivity in beef from cattle younger than 21 months in Japan with that in beef from the United States as assessed by the carcass maturity score

After the detection of the first case of bovine spongiform encephalopathy (BSE) in the United States in December 2003, the Japanese government halted all imports of U.S. beef. The BSE risk in beef is partly dependent on the slaughter age of the cattle from which it is derived. In Japan, all cattle 21 months old and older are screened using a rapid diagnostic test, while in the United States, routine BSE testing is not done at any age of slaughter cattle. In the United States, there is no nationally mandated cattle identification system that enables cattle younger than 21 months to be identified. Therefore, all beef potentially produced for export to Japan must be from cattle that are age verified as younger than 21 months old or be classified by the U.S. Department of Agriculture using a carcass maturity score, which in turn is related to the age of the animal from which the beef is derived. After consulting the Food Safety Commission, the Japanese government decided on 12 December 2005 to allow importation of beef from the United States derived from cattle with a carcass maturity score of < or =A40 and from which specified risk materials are removed. In this study, a stochastic model was used to simulate the interval of time from slaughter to the predicted clinical onset of BSE in an infected animal. A simulation result, based on the assumption that the BSE prevalence is equivalent in the two countries, revealed that there was no increased risk of BSE infectivity in beef coming from carcasses with a maturity score of < or =A40 in the United States, compared with beef from cattle younger than 21 months slaughtered in Japan.     

Comparative studies of a real-time PCR method and three enzyme-linked immunosorbent assays for the detection of central nervous system tissues in meat products

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.     

DETECTION OF CENTRAL NERVOUS SYSTEM TISSUES AS BOVINE SPONGIFORM ENCEPHALOPATHY SPECIFIED RISK MATERIAL IN PROCESSED AND RAW MEAT PRODUCTS IN TURKEY

Spinal cord and brain tissues have the highest importance in the transfer of bovine spongiform encephalopathy to humans. The European Union issued the complete removal of spinal cord and brain tissues from meat products. In Turkey, regulation on meat products is applied by government veterinarians. But there are some questions on the effective application of the regulation, especially in some private slaughterhouses. This study was designed to reveal the mixing of these tissues to meat products sold in the Akdeniz region of Turkey. For this purpose, a total of 471 meat product samples were tested. Two semiquantitative enzyme‐linked immunosorbent assay kits (Ridascreen Risk Material and Risk Material 10/5 [R‐biofarm GmbH, Darmstadt, Germany]), which detect glial fibrillary acidic protein as marker, were used. In the tests, standard controls 0, 0.2, 1.0 and 2.0%, and 0, 0.1, 0.2 and 0.5% were practiced, respectively. Of the 220 processed meat sampled, 33 samples (15.0%) gave an optical density (OD) equal to or higher than standard 0.2%. Of the 251 raw meat sampled, 54 samples (21.51%) gave an OD equal to or higher than standard 0.1%. It was concluded that mixing of central nervous system tissue to the meat products still happens, and more effective control measurements need to be developed and applied in Turkey.     

Some aspects of the effectiveness of stunning in sheep by the captive bolt

The effects of penetration depth and the velocity of the bolt on the onset of unconsciousness was investigated in twenty-four sheep of about 45 kg liveweight. The sheeps' state of consciousness was judged from the electrocorticograms recorded via electrodes surgically pre-implanted frontally on the right side of the head. Sixteen sheep were trephined. A commercial captive bolt pistol and an experimental manual bolt, both with variable penetration depths, were used to represent high and low velocity stunning. Stunning by destroying only the cortex or by only penetrating the skull when the experimental bolt or captive bolt pistol, respectively, were used, did not produce unconsciousness. Unconsciousness was obtained after destroying the cortex and deeper parts of the brain with the experimental bolt (low velocity) or after destroying the cortex (and deeper parts of the brain by shockwaves) when using a captive bolt pistol (high velocity). To produce unconsciousness the captive bolt should have sufficient energy and length after penetration of the skull to crush the cortex and deeper parts of the brain, either directly or by shockwaves.