Specified Risk Material Removal Practices Can We Reduce the BSE Hazard to Human Health

8/11/2019 Specified Risk Material Removal Practices Can We Reduce the BSE Hazard to Human Health

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Speci ed risk material removal practices: Can we reduce the BSE hazard to humanhealth?D. Pitardia, D. Meloni a, * , C. Maurella a, D. Di Vietroa, L. Nocillaa, A. Piscopob, E. Pavolettic, M. Negrod,M. Caramellia, E. Bozzettaaa Istituto Zoopro lattico Sperimentale del Piemonte, Liguria e Valle d Aosta, Via Bologna 148, 10154 Torino, Italyb Azienda Sanitaria Provinciale 1AG, Viale della Vittoria 321, 92100 Agrigento, Italyc Azienda Sanitaria Locale VC, Via Benadir 35, 13100 Vercelli, Italyd Azienda Sanitaria Locale CN1, Corso Francia 12, 12100 Cuneo, Italy

a r t i c l e i n f o

Article history:Received 14 March 2012Received in revised form30 July 2012Accepted 21 August 2012

Keywords:BSEvCJDSpinal cordSRM contaminationAlternative slaughter practices

a b s t r a c t

Following the bovine spongiform encephalopathy (BSE) epidemics across Europe in the early 1990s, theremoval of designated BSE speci ed risk material (SRM) became mandatory to minimize the risk of consumer s exposure to the infectious agent. Despite this preventive measure, cross-contamination of edible meat with SRM can occur during conventional slaughter, primarily by carcass splitting, captivebolt stunning, and head removal. BSE-affected subjects accumulate the pathological feature in the centralnervous system (CNS). Currently, there are no markers that can identify the presence of SRM in meat asa whole. Nevertheless, some assays are able to detect traces of CNS, hence this parameter is of ciallytaken as indicative of SRM contamination.

In this two-stage study, we carried out a survey to estimate the prevalence of carcass contamination attwo slaughterhouses, one large and the other medium-sized; we then compared three different methods(conventional vs. suction vs. water-jet) for spinal cord removal employed at the large slaughterhouse toassess their performance in preventing the spread of central nervous tissue (CNT) over the carcass.

The prevalence of carcass contamination was 68.4%. Compared to the other two spinal cord removaltechniques (conventional and suction), the water-jet system was associated with less CNS contamination(62 vs. 60 vs. 36%, respectively; P 0.0047).

2012 Published by Elsevier Ltd.

1. Introduction

Transmissible spongiform encephalopathies (TSEs), also termedprion diseases, are fatal, neurodegenerative disorders, whichinclude Creutzfeldte Jakob disease (CJD) in humans, bovine spon-giform encephalopathy (BSE) in cattle, and scrapie in sheep andgoat. BSEwasdescribed for the rst timein 1987 asa new disease incattle (Brown, Will, Bradley, Asher, & Detwiler, 2001; Bruce et al.,1997). Ten years later in the U.K. a previously unseen fatal neuro-degenerative disease resembling CJD was recognized in humans.The epidemiological trend of both diseases and further scienti cevidence unequivocally established the link between BSE and thenew variant of CJD (vCJD) and the risk to humans posed by theconsumption of infected bovine tissues ( Brown et al., 2001; Bruceet al., 1997).

In BSE-infected cattle, infection source accumulates in speci corgans (brain, spinal cord, tonsils, distal ileum, dorsal root ganglia,trigeminal ganglia and eyes) referred to as speci ed risk materials(SRM) (Bowling et al., 2007). Under the provisions of Regulation(EC) No 999/2001 to protect public health, these organs havebeen banned from entering the human food chain in all EuropeanUnion Member States.

Nevertheless, during slaughtering practices, both brain andspinal cord tissues can contaminate carcasses and, hence, enter thefood chain (Bowling et al., 2007). Conventional slaughter tech-nology continues to present signi cant opportunities for centralnervous system (CNS) material to contaminate or cross-contaminate meat. Captive bolt stunning, improper removal of SRM, carcass washing and splitting represent the pointed routes of contamination ( Takada, Horiuchi, Sata, & Sawada, 2008; Troeger,2004). Depending on the stunning devices, CNT may enter thebloodstream and disseminate throughout the carcass ( Anil, Love,Helps, & Harbour, 2002; Anil et al., 1999; Coore et al., 2004; Cooreet al., 2005; Lim, Erwanto, & Lee, 2007; Love et al., 2000;

* Corresponding author. Tel.: 39 (0) 11 2686347; fax: 39 (0) 11 2686322.E-mail address: [email protected] (D. Meloni).

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0956-7135/$ e see front matter 2012 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.foodcont.2012.08.005

Food Control 30 (2013) 668 e 674
mailto:[email protected]://www.sciencedirect.com/science/journal/09567135http://www.elsevier.com/locate/foodconthttp://dx.doi.org/10.1016/j.foodcont.2012.08.005http://dx.doi.org/10.1016/j.foodcont.2012.08.005http://dx.doi.org/10.1016/j.foodcont.2012.08.005http://dx.doi.org/10.1016/j.foodcont.2012.08.005http://dx.doi.org/10.1016/j.foodcont.2012.08.005http://dx.doi.org/10.1016/j.foodcont.2012.08.005http://www.elsevier.com/locate/foodconthttp://www.sciencedirect.com/science/journal/09567135mailto:[email protected]


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on raw meat, meat products and on contaminated surfaces. In orderto facilitate application of the test for screening purposes, inprevious studies we validated its qualitative use by plotting an ROCcurve to set a useful cut-off value.

A volume of 50 ml of standards (0, 0.1, 0.2 and 0.4% CNSconcentration) in duplicate and 50 ml of samples were distributedinto wells originally coated with the anti-GFAP primary antibody.Next, 50 ml of conjugate were added to each well, and the test platewas incubated at room temperature for 10 min. After three-stepwashing, 100 ml of substrate/chromogen were added prior toincubation for 5 min. The reaction was stopped by adding 100 ml of blocking solution. The plates were then read at 450 nm (Tecan,SUNRISE, Grder, Salzburg, Austria) to obtain the optical density foreach well. The RIDA SOFT Win software (R-Biopharm AG) allowsautomated data retrieval from the reader and calculation of the

results by linear regression. In caseof positive results, samples wereimmediately retested in duplicate.

3. Statistics

The sample size for determining the prevalence level forcontamination at the slaughterhouse was calculated by taking intoaccount the results of the previous study by the same work group(Bozzetta et al., 2006). A prevalence of contamination of 15%, a 95%con dence level, and an accepted error of 4% were assumed toestimate the sample as representative of the two abattoirs.

The sample size necessary to evaluate differences among thethree removal methods was obtained by tting the Kastenbaum,Hoel and Bowman tables to our data Kastenbaum, Hoel e Bowmantables for ANOVA (Woolson & Clarke, 2002). Data distribution was

Fig. 2. Spinal cord removal with the water-jet system; when the carcass is located in the splitting area (A) the sacrum is split along its length to display the vertebral channel (B),a hose pipe connected to a tap is inserted into the channel (C); when the faucet is turned on, the water pressure causes the spinal cord to leak out through the atlas (D).

Fig.1. Spinal cord removal by vacuum suction. After the carcass is decapitated, a guide tube is inserted through which a PVC hose is advanced caudally into the vertebral channelwith caution, the spinal cord is then extracted under vacuum.

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evaluated by means of a test for normality based on skewness andkurtosis. As the data obtained were not normally distributed, theKruskale Wallis equality-of-populations rank test was performed toverify the key differences between contamination levels associatedwith the three SRMremoval techniques. The data were entered intoan ad hoc database and analysed using Stata 11 SE software.

4. Results

Inthe rststageofthisresearchwecarriedoutasurveyinordertoevaluatethelevelofCNTcontaminationusingconventionalslaughter

technique. A total of 412 bovine carcasses were investigated after

splitting and spinal cord removal in two slaughterhouses; one largeandtheother medium-sized.Sampleswere de nedaspositiveifCNStissue was detected at a concentration 0.049%.

The lowest level of CNT contamination was detected in thelarge slaughterhouse applying the suction technique; samplestested positive in 130 out of 216 carcasses (60.2%, 95% CI [con-dence interval], 53.3e 66.8). In the medium-sized one, the spinalcord was manually removed and 152 out of 196 carcasses testedpositive (77.6%, 95% CI, 71e 83.2). Table 1 summarizes the resultsstrati ed by contamination level. Lastly, the survey data assessedwere used to establish the overall prevalence of CNT contamina-

tion connected with the conventional slaughter practice: an

Fig. 2. (continued ).

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overall prevalence of CNT contamination of 68.4% (95% CI, 53e 83)was shown.

In the second stage of this study we compared the conventionalpractice with two alternative methods by which the SRM isextracted before the carcass is split. The sample size was 50carcasses for each SRM removal method.

The comparative study showed a CNS contamination of 62%(95% CI, 47.2e 75.3) associated with the conventional technique,60% (95% CI, 45.2e 73.6) with the suction technique, and 36% (95%CI, 22.9e 50.8) with the water-jet system ( Fig. 4). The differenceamong the three methods appeared to be signi cant (P 0.0047)(Table 2). The percentages of contamination strati ed according tothe spinal cord removal technique and the relative level of CNS

contamination are shown in Table 3.

5. Discussion

Numerous reports have linked the presence of critical points inconventional slaughter practices with the subsequent contamina-tion of carcasses by SRM (Anil et al., 2002, 1999; Bowling et al.,2007; Coore et al., 2004, 2005; Helps et al., 2004, 2002; Kaleet al., 2008; Lim et al., 2007; Love et al., 2000; Prendergast et al.,2003; Takada et al., 2008). Carcass splitting has been identi ed asthe key stage for contamination by SRM, due to the mixture of sawing residues and rinsing water, or sawing sludge , that collectsin the saw housing ( Bowling et al., 2007; Helps et al., 2004, 2002;Kale et al., 2008; Prendergast et al., 2003; Ramantanis, 2006;Takada et al., 2008; Troeger, 2004). To overcome this problem,several alternative slaughter methods were developed by whichthe SRM is extracted prior to splitting (Piscopo, 2001; Ramantanis,2006; Troeger, 2004).

The aim of our study was to estimate the prevalence of SRMcontamination of carcasses on the medial split surface of theparavertebral muscles of the cervical region following the use of the conventional splitting method. This was done in order to

evaluate whether the CNS contamination was less with twoalternative techniques in which the carcass is split after spinalcord removal.

Table 1Conventional method e positive samples strati ed according to contamination leveland type of abattoir.

CNS contamination Large abattoirno. positivesamples (%)

Medium abattoirno. positivesamples (%)

Low ( 0.049 and < 0.2) 53 (40.8) 105 (69.1)Moderate ( 0.2 and < 0.4) 31 (23.8) 35 (23)High ( 0.4) 46 (35.4) 12 (7.9)Total positive samples 130 (60.2) 152 (77.6)

60%

36%

62%

64%

38% 40%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Tradit ional Suct ion Water-jet

Negative samples Positive samples

Fig. 4. The histogram shows the percentages of positive and negative samples groupedby method.

Fig. 3. Schematic diagram of the area swabbed on the medial surface of the splitcarcass.



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The ELISA RRM 10/5 was applied as a qualitative assay. Theestimated cut-off value excludes a possible reactivity related to theperipheral nerve counterpart ( Bozzetta et al., 2006).

In previous studies, assessment of the contamination levels indifferent sampling areas of the carcass indicated that CNScontamination is greater on the internal surfaces along the cutvertebral surface, where the lumbar and cervical regions werefound to be the most contaminated ( Helps et al., 2002; Prendergastet al., 2003). Accordingly, we selected as the target sampling areathe paravertebral muscles of the cervical region.

The highest percentage of CNS contamination was associatedwith the conventional slaughtering method. When the positivesamples werestrati ed by type of abattoir and contamination level,the percentage of positive samples was less for the large abattoir.This difference might have been due to generally better handlingtechniques or the suction device used to eliminate the spinal cordafter carcass splitting, which might require less carcass manipula-tion than manual removal as employed in the medium-sizedfacility.

When compared against the conventional method, the twoalternative slaughter techniques were associated with a lower levelof CNS contamination on paravertebral meat. However, contami-nation after spinal cord removal by suction was only slightly lessthan the one obtained using the traditional method (60 vs. 62%,respectively). This maybe explained by the fact that thespinal cord,being encased by the vertebral bones, cannot be entirely removed,especially from the lumbar tract, due to the still imperfect set up of

the device. Furthermore, the technique requires a certain degree of dexterity and skill in inserting and advancing the hose into thevertebral channel, which may not always be realistic under eldconditions. Other problems were related to the frequent hoseclogging with spinal cord tissue and the occurrence of vertebralbone breaks or dislocations.

On the other hand, the spinal cord removal with the water-jetwas associated with a 30% reduction in CNS contamination: 89%of positive samples had a low level of CNS contamination. Thetechnique is quick and easy to be applied, it doesn t require anydedicated training. When the carcass is located in the splitting areathe sacrum is cut along its length to display the vertebral channel;a hose pipe connected to a water tap is inserted into the vertebralchannel and subsequently the faucet is turned on. The water

pressure causes the SRM to leak out immediately through the atlas.The method allowed the complete extraction of the spinal cordfrom each carcass and the results showed that the removal of the

whole spinal cord before splitting prevents the spread of thecontamination on the carcass surface.

The level of CNS contamination still present on carcasses treatedby this method was further investigated. Since previous studiesdemonstrated that the splitting saw plays an important role indisseminating the CNS tissue from one carcass to the next ( Helpset al., 2004), the rst and the last-slaughtered carcasses were dayby day sampled and analysed after applying the water jet treat-ment, as to monitor the spread of the contamination. Under suchsimulated abattoir conditions, the remaining contamination ratewas found out to be effectively related to the scatter of the CNStissue remaining in the saw housing from one carcass to another(data not shown). Dif culties resulting from occasional breaks ordislocation of vertebral bones have also been reported with thismethod.

6. Conclusions

According to EC Regulation 999/2001, speci ed risk materialsand possibly CNS tissues have been banned from entering thehuman food chain, as these tissues harbour the highest level of infection. Nevertheless, CNS tissues can still contaminate carcassesduring slaughtering. This could mean that if an animal with sub-clinical BSE or an undiagnosed infected animal is slaughtered, theinfectious agent (PrPsc) could spread over the carcass, potentiallyexposing consumers to infection.

Despite efforts to control contamination by CNS tissue, high-volume abattoirs in Europe are reluctant to adopt new technolo-gies purportedly because of increased production costs and time.This could be partially justi ed by the wide use of highly sensitiveand speci c BSE rapid tests for screening purposes.

In light of the encouraging trends in the control of BSE, theEuropean Commission has outlined possible future changes to EUmeasures on TSEs, introducing a gradual risk-based lift of the feedban and an increase of the age limit for testing ( EuropeanCommission, Brussels, 16.7.2010). In this context, the develop-ment of alternative strategies, and their application to slaughtermethods, could inform risk-based policies. This study incontro-vertibly shows that an alternative slaughter practice can reduce theCNS contamination of meat products and help to protectconsumers against the risk of exposure to BSE.

Acknowledgements

This study was funded by the Italian Ministry of Health (ProjectIZSPLV 03/08RC).

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Specified Risk Material Removal Practices Can We Reduce the BSE Hazard to Human Health