NR AAHJ

AU Anil,M.H.; Love,S.; Williams,S.; Shand,A.; McKinstry,J.L.; Helps,C.R.; Waterman-Pearson,A.; Seghatchian,J.; Harbour,D.A.

TI Potential contamination of beef carcases with brain tissue at slaughter

QU The Veterinary Record 1999 Oct 16; 145(16): 460-2

PT journal article

VT EXPERIMENTAL strain typing of variant Creutzfeldt-Jakob disease (vCJD) has shown that the transmissible agent responsible for this disorder is identical to that in bovine spongiform encephalopathy (BSE), providing further evidence to support the hypothesis that exposure to the BSE agent, presumably through the diet, is the cause of vCJD (Ironside 1998). Most cattle are stunned with a captive bolt gun (CBG) before slaughter and because of the suggested link between BSE and vCJD, the possibility that the use of CBGs may contaminate the car-case with brain tissue has raised concern for public health.
Garland and others (1996) previously reported the finding, at necropsy, of brain tissue in the lungs of cattle slaughtered in the USA after being stunned with a pneumatically activated, penetrating CBG that introduced air intracranially. However, the validity and relevance of the Garland report was questioned (Taylor 1996) and similar studies carried out on 210 cattle in UK abattoirs failed to confirm this observation (Munro 1997). The UK Meat and Livestock Commission issued a press release on September 29, 1996, stating that the report had no relevance to the meat industry in Britain (Anon 1996). However, relatively crude and insensitive procedures were used in these studies for the detection of central nervous system ( CNS) material. Concern, therefore, remains that the stunning and slaughter procedures that are prevalent in Europe and the USA may possibly, if used in an animal that had BSE, cause contamination of the edible parts of the carcase with brain tissue containing high titres of infectivity, and therefore expose the consumer to the risk of developing vCJD. In this study, different CBGs and slaughter procedures were investigated to evaluate whether or not they caused the entry of brain tissue into the jugular venous blood. The bovine heart continues to pump for several minutes following the use of a CBG, during which time any CNS material that enters the jugular venous blood may be disseminated throughout the body.
Intravenous catheters were placed in the animals' jugular vein for the injection of anaesthetic agent and the animals were moved into a stunning box. They were then anaesthetised before slaughter, with intravenous ketamine (1 mg/kg bodyweight) and xylazine (0.6 mg/kg bodyweight). Following induction of anaesthesia, the animals were tipped out of the stunning box. Anaesthesia was maintained with a continuous infusion of a mixture of xylazine (0.01 per cent) and ketamine (0.1 per cent) in guaiphenesin (5 per cent) at a rate of 20 ml/minute per animal, for cardiovascular stability. Foley catheters were introduced into the jugular veins, a prestun blood sample was taken, then the catheters were inflated and the animals were immediately stunned. Sixty animals in total were used. Treatments included the use of penetrating and non-penetrating CBGs, with or without air injection and with or without pithing, that is destruction of the brain stem and spinal cord by a flexible rod inserted through the hole made by the captive bolt. The makes and models of CBGs tested were: a pneumatically operated air injection penetrating CBG (no pithing required due to air injection into spinal canal) (Hantover); a penetrating cartridge operated conventional CBG (with or without subsequent pithing), known as a Cow Puncher (Accles and Shelvoke); and a non-penetrating cartridge operated CBG (therefore no pithing), known as a Cash Knocker (Accles and Shelvoke). The shooting position for each gun was the intersection between two lines extending from the back of the eye to the contralateral horn bud. In the 60 seconds following stunning, the blood draining from the jugular catheters was collected in 250 ml citrated bottles, each bottle receiving the jugular venous output over a 10-second period. Following sample collection, each animal was hoisted up and exsanguinated by conventional procedures.
A 1 ml subsample of whole blood was taken from each aliquot collected over each 10-second period, for use in capture enzyme-linked immunosorbent assays (ELISAs) for syntaxin 1-B, an integral membrane protein exclusively and abundantly expressed in nervous tissue, and annexin V, an endogenous cytoplasmic protein with anticoagulant activity. The release of soluble and microvesicle-bound annexin V in plasma has previously been used as a marker of cellular damage (Krailadsiri and others 1997) including that involving the CNS (Woolgar and others 1990). The remaining blood was centrifuged and the buffy coat removed, fixed in an equal volume of 20 per cent formalin, pelleted by further centrifugation and embedded in paraffin wax; pilot studies on deliberately contaminated blood samples showed that brain tissue separated with the bufly coat. Sections at multiple 1evels were cut for conventional histology and for immunostaining with antibody to S100ß protein (Dako). S100ß is a calcium binding protein abundantly, though not exclusively, expressed in nervous tissue (Barger and van Eldik 1992). In pilot studies, the method was found to be reliably sensitive enough to detect 50 mg or more of brain tissue/100 ml whole blood. Antibodies to several other neuronal and glial antigens ( including neuron-specific enolase, microtubule associated protein 2, synaptophysin and glial fibrillary acidic protein) were tested, but immunocytochemistry for S100ß protein gave the most consistent results.
A capture ELISA for syntaxin 1-B was developed. ELISA plates were coated with 50 µl mouse monoclonal anti-syntaxin (clone 78.2; Synaptic Systems) at 1 µg/ml and incubated overnight at 4°. The plates were washed and non-specific protein binding was blocked by adding 200 µl phosphate buffered saline (PBS)/0.05 per cent Tween 20/5 per cent non-fat, dried, skimmed milk (blocking solution) at room temperature for one hour. The plates were again washed and 50 µl of each sample, comprising 25 µl whole blood mixed with 25 µl cell lysis solution, was added to each well and incubated at room temperature for two hours. After a further wash, 50 µ1 of blocking solution containing a 1:1000 dilution of rabbit anti-syntaxin polyclonal serum (Synaptic Systems) was added per well and incubated at room temperature for one hour. The plates were washed and 50 µl of blocking solution containing 1 µg/ml alkaline phosphatase-conjugated donkey anti-rabbit immunoglobulin G (IgG) (Jackson Immunoresearch Laboratories) was added to each well and incubated at room temperature for one hour. After a final wash, 50 µl of 1 mg/ml p-nitrophenol phosphate was added to each well to allow development of the ELISA over 24 hours. The plates were then read at 405 nm to obtain the optical density of each well. Each ELISA plate also contained a row of wells with serial dilutions of recombinant purified syntaxin produced in Escherichia coli. These allowed the generation of a standard curve and the quantification of the sample results. The sensitivity of this assay was determined to be 1 to 2 ng syntaxin 1-B/ml whole blood.
A capture ELISA kit (Stago) was used according to the manufacturer's protocol for quantitating annexin V, as described previously by Krailadsiri and others (1997). The assay was performed on a 1:5 dilution of plasma and the results were calculated using a standard curve. The sensitivity of the ELISA for annexin V was 0.125 ng/ml.
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FIG 1 : Jugular blood samples containing (a) morphologically obvious brain tissue (x 135) and (b) fragments (arrows) which were difficult to discern in haematoxylin and eosin preparations (x 70) but (c) stained strongly for S100ß protein (arrows) (x 70)
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FIG 2: Venous blood syntaxin 1-B levels in two animals stunned with a penetrating captive bolt gun (cBG) (solid line) and a conventional CBG followed by pithing (interrupted line)
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Multiple fragments of brain tissue were detected in the jugular venous blood of four of the 15 cattle slaughtered after use of the pneumatically operated air injection penetrating CBG. Brain tissue fragments were detected by microscopy and immunocytochemistry of buffy coat material and by syntaxin 1-B ELISA of whole blood. Plasma derived from blood samples from the same animals was also positive by ELISA for annexin V. Jugular venous blood from another of the 15 animals slaughtered after use of the above penetrating CBG yielded positive results in the syntaxin 1 -B and annexin V assays, although brain material was not detected in this animal by microscopy. Brain tissue was also found in the venous blood of one of 16 cattle slaughtered by a conventional penetrating CBG with subsequent pithing (used in 70 per cent of UK abattoirs) (Anon 1997); the brain tissue was detected on microscopy and immunocytochemistry for S1OOß protein, and the blood contained elevated levels of syntaxin 1 -B and annexin V. No brain tissue or elevation of syntaxin 1 -B or annexin V was detected in the blood from the 15 cattle slaughtered after the use of a conventional Cow Puncher CBG (Accles and Shelvoke) without pithing or from 14 animals slaughtered after the use of a mushroom head, non-penetrating CBG (Cash Knocker; Accles and Shelvoke).
The embolic brain tissue was recognisable as such morphologically, although its detection was facilitated by immunocytochemistry for S100ß protein (Fig 1). This revealed many fragments of varying size, some less than 50 µm in diameter. Fig 2 shows the time course of detection of brain tissue, by capture ELISA for syntaxin, in sequentially sampled jugular venous blood taken from animals after the use of a penetrating CBG (Hantover) or a conventional CBG (Cow Puncher; Accles and Shelvoke) followed by pithing. The time course of syntaxin elevation corresponded to that of annexin V which was detected in the jugular blood samples within 20 to 40 seconds of stunning. The appearance of annexin V in these samples also coincided with a prolongation of tissue factor/calcium induced coagulation (results not shown).
The demonstration of a causal relationship between the agent of BSE and vCJD (Ironside 1998), and the report of brain emboli in the lungs of cattle slaughtered with a penetrating CBG (Garland and others 1996) caused concern that the removal of specified risk materials from slaughtered animals may not be sufficient to completely remove the risk of transmission of BSE prions to humans through meat consumption. These results suggest that there may be a potential risk of embolic dissemination of brain tissue with the use of a pneumatically operated air injection gun and show, in addition, that neuroembolism may possibly occur with use of a conventional penetrating CBG followed by pithing. The emboli are detectable in jugular venous blood within 30 seconds of stunning and may already have passed into and, possibly, through the lungs before exsanguination is carried out (at about 90 seconds). These findings are in keeping with observations of embolic brain tissue in the lungs of human victims of head injury (McMillan 1956, Hatfield and Challa 1980) and of elevated neuron-specific enolase and BB creatine kinase in the serum (Skogseid and others 1992). Although the question as to whether or not emboli reach the arterial circulation and are deposited in edible tissues needs further detailed investigation, it is noteworthy that the showers of embolic brain tissue include many fragments of sufficiently small size to be capable, in principle, of passing through the pulmonary capillary bed.
In conclusion, the results confirm the potential risk of haematogenous dissemination of CNS tissue with the use of a pneumatically operated gun and show, in addition, that neuroembolism may also occur when use of a conventional penetrating CBG is followed by pithing. However, the question as to whether emboli can reach arterial circulation and be deposited in edible tissues needs further investigation. Further research is required and possibly a review of procedures for the stunning and slaughter of cattle in UK abattoirs.
ACKNOWLEDGEMENTS
This work was funded by the Ministry of Agriculture, Fisheries and Food (SE 1830) The capture ELiSA for syntaxin 1-B was developed as part of an EC-funded shared cost project (CT97-3301).
References
ANON (1996) Press release 66/96. Meat and Livestock Commission
ANON (1997) Abattoir welfare survey, November. Meat Hygiene Service
BARGER, S. w. & VAN ELDIK, L. J. (1992 ) S100ß stimulates calcium fluxes in glial and neuronal cells. Journal of Biological Chemistry 267, 9689-9694
GARLAND, T., BAUER, N. & BAILEY, M. (1996) Brain emboli in the lungs of cattle after stunning. Lancet 348, 610
HATFIELD, S. & CHALLA, V. R. (1980) Embolism of cerebral tissue to lungs following gunshot wound to head. Journal of Trauma 20,353-355
IRONSIDE, J. W. (1998) Neuropathological findings in new variant CJD and experimental transmission of BSE. FEMS Immunology and Medical Microbiology 21, 92-95
KRAILADSIRI, P., SEGHATCHIAN, J., AMIRAL, J., VISSAC, A. M. & CONTRERAS, M. (1997) Annexin V: a new marker of platelet storage lesion. Transfusion Science 18, 223-226
McMILLAN, J. B. (1956) Embolism of cerebral tissue ih lungs following severe head injuri. American Journal of Pathology 32, 405-415
MUNRO, R. (1997) Neural tissue embolism in cattle. Veterinary Record 140, 536
SKOGSEID, I. M., NORDBY, H. K., URDAL, R, PAUS, E. & LILLEASS, E (1992) Increased serum creatine kinase BB and neuron specific enolase following head injury indicates brain damage. Acta Neurochirurgica 115, 106-111
TAYLOR, K. C. (1996) Brain emboli in the lungs of cattle. Lancet 348, 749
WOOLGAR, J. A., BOUSTEAD, C. M. & WALKER, J. H. (1990) Characterization of annexins in mammalian brain. Journal of Neurochemistry 54, 62-71

IN Die meisten britischen Rinder werden vor dem Schlachten mit einem Bolzenschußapparat betäubt, aber ihr Herz schlägt danach noch einige Minuten. Garland et al. berichteten 1996 über Hirngewebe in den Lungen von in den USA geschlachteten Rindern [GWF]. Diese waren vor dem Schlachten mit einem pneumatischen Bolzenschußapparat betäubt worden, der Luft in den Schädel schießt [GWF]. Taylor hatte (allerdings ohne gute Gründe) die Darstellung von Garland et al. in Zweifel gezogen [ANCE] und weitere Tests angekündigt. Diese Studie mit relativ unempfindlichen Methoden an lediglich 210 britischen Rindern wurde dann ohne entsprechende Befunde und seltsamerweise allein von Munro publiziert [ANCF]. Dennoch kam die britische Meat and Livestock Commission bereits mit einer Presseerklärung vom 29.9.1996 zu dem Schluß, der Garland-Bericht habe keine Bedeutung für die britische Fleischindustrie.
Zur Überprüfung der unklaren Situation plazierten Anil et al. intravenöse Katheter in die vom Gehirn kommenden Halsvenen (Drosselvenen). Diese wurden zunächst zur intravenösen Betäubung mit Ketamin (1 mg/kg Körpergewicht) und Xylazin (0,6 mg/kg Körpergewicht) benutzt. Die Betäubung wurde durch eine kontinuierliche Infusion mit 20 ml/Minute Xylazine (0,01 %) and Ketamine (0,1 %) in Guaiphenesin (5 %) aufrecht erhalten. Nach Einsetzen der Betäubung wurden bei den insgesamt 60 Tieren unterschiedliche Bolzenschußapparate eingesetzt und während der hierauf folgenden 60 Sekunden wurden von jedem Tier etwa 6 mal 250 ml Blut durch die Katheter aufgefangen. Verglichen wurden ein nicht in das Gehirn eindringendes Gerät (Cash Knocker von Accles and Shelvoke), ein in das Gehirn eindringendes Gerät (Cow Puncher von Accles and Shelvoke) und der pneumatische "The Knocker" der Firma Hantover, welcher das Gehirn durch eindringende Luft zerreißt. Bei einem Teil der mit dem Cow Puncher tödlich verletzten Tiere wurde durch den Bolzenkanal hindurch mit einer flexiblen Rute Hirnstamm und Rückenmarkansatz zerstört. Man nennte dies "pithing". Danach wurden die Tiere normal ausgeblutet.
In den während jeweils etwa 10 Sekunden gewonnenen Blutproben wurde per ELISA nach den Hirnmarkerproteinen Syntaxin 1-B und Annexin V gesucht. Mit einem speziell entwickelten ELISA konnten die Autoren 1-2 ng Syntaxin 1-B pro ml Gesamtblut nachweisen. Mit einem kommerziellen ELISA erreichten sie eine Empfindlichkeit von 0.125 ng/ml für den Nachweis von Annexin V. Die restlichen Blutproben wurden zentrifugiert und die aus Leukozyten und Thrombozyten bestehende Schicht (buffy coat) zwischen Serum und Erythrozyten wurde abgenommen und durch Zugabe der gleichen Menge 20% Formalin fixiert. Pilotstudien hatten gezeigt, dass Hirnmaterial nach der Zentrifugation in der buffy coat konzentriert war. Die Fixierungssuspension wurde abzentrifugiert und die festen Bestandteile wurden danach in Paraffinwachs eingebettet. In Dünnschnitten dieser Präparate wurde histologisch und immunhistologisch nach Hirnmaterial gesucht.
Mikroskopisch und immunzytochemisch waren viele, zum Teil weniger als 50 µm dicke Hirnstücke im aufgefangenen Blut von 4 der 15 Rinder erkennbar, die mit dem pneumatischen Bolzenschußapparat der Firma Hantover betäubt worden waren. Per ELISA gelang der Nachweis von Hirnmaterial im Gesamtblut dieser vier und eines weiteren ohne erkennbare Hirnfragmente. Hirngewebe wurde aber auch mikroskopisch, immunzytochemisch und per ELISA im venösen Blut von einem der 16 Rinder gefunden, die mit dem konventionellen invasiven Bolzenschußapparat und anschließendem pithing betäubt wurden, wie es in 70% der britischen Schlachthöfen üblich ist. Im Blut der 15 mit dem konventionellen invasiven Bolzenschußapparat ohne anschließendes pithing sowie in den 14 mit dem nichtinvasiven Bolzenschußapparat betäubten Rinder fand man keine Hinweise auf Hirnmaterial.
Die ELISA-Auswertung der 6 Blutfraktionen zeigte, dass das Hirngewebe im Wesentlichen binnen 30 Sekunden die Halsvenen passiert und genügend Zeit hat, innerhalb des 90 Sekunden dauernden Ausblutens in die Lunge und vermutlich sogar durch sie hindurch zu gelangen. Die kleinsten Hirnpartikel sind jedenfalls klein genug, um die Lunge zu passieren.

MH *Abattoirs; Animal; Brain/pathology; Cattle; Cattle Diseases/*transmission; Encephalopathy, Bovine Spongiform/*transmission; Enzyme-Linked Immunosorbent Assay; *Food Contamination; Food Handling/methods; Jugular Veins; Meat/*standards; Support, Non-U.S. Gov't

AD M.H.Anil, DVM, PhD, J.L.McKinstry, HNC, A.Waterman-Pearson, PhD, FRCVS, C.R.Helps, PhD, D.A.Harbour, PhD, Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS4O 5DU
S.Love, PhD, FRCP, FRCPath,
S.Williams, HNC, A.Shand, BA, Department of Neuropathology, Frenchay Hospital, Bristol BS16 iLE
J.Seghatchian, MD, PhD, National Blood Service, London and SE Zone, Colindale Avenue, London NW9 SBG

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