Specied risk material removal practices: Can we reduce the BSE hazard to human

health?
D. Pitardi
a
, D. Meloni
a,
*
, C. Maurella
a
, D. Di Vietro
a
, L. Nocilla
a
, A. Piscopo
b
, E. Pavoletti
c
, M. Negro
d
,
M. Caramelli
a
, E. Bozzetta
a
a
Istituto Zooprolattico Sperimentale del Piemonte, Liguria e Valle dAosta, Via Bologna 148, 10154 Torino, Italy
b
Azienda Sanitaria Provinciale 1AG, Viale della Vittoria 321, 92100 Agrigento, Italy
c
Azienda Sanitaria Locale VC, Via Benadir 35, 13100 Vercelli, Italy
d
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 2012
Received in revised form
30 July 2012
Accepted 21 August 2012
Keywords:
BSE
vCJD
Spinal cord
SRM contamination
Alternative slaughter practices
a b s t r a c t
Following the bovine spongiform encephalopathy (BSE) epidemics across Europe in the early 1990s, the
removal of designated BSE specied risk material (SRM) became mandatory to minimize the risk of
consumers 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, captive
bolt stunning, and head removal. BSE-affected subjects accumulate the pathological feature in the central
nervous system (CNS). Currently, there are no markers that can identify the presence of SRM in meat as
a whole. Nevertheless, some assays are able to detect traces of CNS, hence this parameter is ofcially
taken as indicative of SRM contamination.
In this two-stage study, we carried out a survey to estimate the prevalence of carcass contamination at
two 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 to
assess 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 removal
techniques (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 termed
prion diseases, are fatal, neurodegenerative disorders, which
include CreutzfeldteJakob disease (CJD) in humans, bovine spon-
giform encephalopathy (BSE) in cattle, and scrapie in sheep and
goat. BSE was described for the rst time in 1987 as a newdisease in
cattle (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 scientic
evidence unequivocally established the link between BSE and the
new variant of CJD (vCJD) and the risk to humans posed by the
consumption of infected bovine tissues (Brown et al., 2001; Bruce
et al., 1997).
In BSE-infected cattle, infection source accumulates in specic
organs (brain, spinal cord, tonsils, distal ileum, dorsal root ganglia,
trigeminal ganglia and eyes) referred to as specied risk materials
(SRM) (Bowling et al., 2007). Under the provisions of Regulation
(EC) No 999/2001 to protect public health, these organs have
been banned from entering the human food chain in all European
Union Member States.
Nevertheless, during slaughtering practices, both brain and
spinal cord tissues can contaminate carcasses and, hence, enter the
food chain (Bowling et al., 2007). Conventional slaughter tech-
nology continues to present signicant opportunities for central
nervous 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 the
bloodstream and disseminate throughout the carcass (Anil, Love,
Helps, & Harbour, 2002; Anil et al., 1999; Coore et al., 2004; Coore
et 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: daniela.meloni@izsto.it (D. Meloni).
Contents lists available at SciVerse ScienceDirect
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j ournal homepage: www. el sevi er. com/ l ocat e/ f oodcont
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) 668e674
Prendergast, Sheridan, Daly, McDowell, & Blair, 2003) or drain from
the animal to slaughtering equipment and abattoir personnel.
Conventional carcass splitting involves separating it into two
sides with a circular band saw down the centre of the vertebral
column. The spinal cord is destroyed and the tissue remnants
spread over the carcass and into the surrounding environment.
Furthermore, since the band saw blade accumulates CNS material
fromthe spinal cord, it acts as a vector of cross-contamination from
one carcass to the next (Bowling et al., 2007; Helps, Fisher, Harbour,
ONeill, & Knight, 2004; Helps et al., 2002; Kale et al., 2008;
Prendergast et al., 2003; Ramantanis, 2006; Takada et al., 2008;
Troeger, 2004). To overcome this problem, some European low-
speed abattoirs extract the spinal cord before the carcass is split.
To verify the presence of CNS material in meat products, several
methods that can detect specic CNS markers have been devel-
oped. Testing methods include tissue dissection and visual
inspection, histological staining (Wenisch, Lcker, Eigenbrodt,
Leiser, & Blte, 1999), immunohistochemistry (IHC) (Kelley,
Hafner, McCaskey, Sutton, & Langheinrich, 2000; Wenisch et al.,
1999), immunochemical assays, i.e., ELISA and Western Blot
methods (Agazzi, Barrero-Moreno, Lcker, von Holst, & Anklam,
2002; Hajmeer, Cliver, & Provost, 2003), quantication of choles-
terol (Lcker et al., 1999), reverse transcription polymerase chain
reaction (RT-PCR) (Schnenbrcher, Gbel, Abdulmawjood, Richt, &
Blte, 2008), and gas chromatographyemass spectrometry (GCe
MS) (Lcker, Biedermann, Lachhab, Truyen, & Hensel, 2004).
Effective immunochemical assays have been recently imple-
mented to detect CNT in meat and meat products. They are in
general highly sensitive, specic and objective, other than relatively
rapid, cheap and easy to apply.
Histological staining and immunohistochemical assays have
been shown to be poor CNS tissue screening tests. Histology is
partially effective on row meat but totally ineffective if applied on
processed meat products subjected to homogenization; moreover
it is highly subjective. Differently, immunohistochemistry revealed
good performance on row, minced and variably heated meat
products. Its to be noted that both techniques are more suitable for
conrmatory roles as require highly trained personnel, expensive
equipment, a long period of time to complete and are limited by the
amount of sample that is viewed for detection (Bowling et al., 2007;
Hossner et al., 2006).
GCeMS and RT-PCR are highly sensitive assays, and they seemto
be useful analytical tools. However, detection of CNS tissue by GCe
MS and RT-PCR is likely impractical on a large scale, everyday basis
due to its high cost, the length of time required to conduct the assay
and the technical expertise required of technicians (Bowling et al.,
2007).
According to Commission Regulation (EC) No 1139/2003,
a sampling plan with an appropriate laboratory test to detect CNS
tissue in bovine head meat must be in place in each Member State.
To full the Regulation, in a previous study the Authors carried out
a survey to quantify the CNT contamination on the head muscles
sourced from the site of dissection of the medulla oblongata at the
level of the foramen magnum and the hole caused by bolt stunning.
As the void of a validated test, we needed to evaluate the validity of
the Ridascreen

Risk Material 10/5 (RRM 10/5) test. In that frame,

we plotted a receiver operating characteristic (ROC) curve to set the
cut-off value of the ELISA test. With this approach the technique
displayed high accuracy and reliability for a cut-off value between
positive and negative samples at a CNS concentration of 0.049%
(Bozzetta et al., 2006).
The aim of the present study was to estimate the prevalence of
CNS contamination of carcasses at two slaughterhouses (one large
and the other medium-sized) and to compare the differences in the
percent of CNS contamination of the carcass surface after the use of
three different spinal cord removal techniques at the large
slaughterhouse. Specically, the conventional practice of sawing
the vertebral column before the removing of the spinal cord was
evaluated versus two alternative methods by which the SRM is
extracted before the carcass is split. The feasibility of the alternative
methods in slaughterhouses, under eld conditions, was investi-
gated as well.
2. Materials and methods
2.1. Prevalence of CNS contamination
The prevalence of CNS contamination by the conventional
technique was estimated from a total of 216 carcasses from a large
slaughterhouse (more than 400 heads per day) and 196 from
a medium-sized one (less than 50 heads per day). In both abattoirs
the animals were stunned using a captive bolt gun, the carcasses
were split with a hand-guided belt-type saw (Jarvis, Suzzara, Italy)
and the spinal cord cut along its length was removed fromeach side
of the carcass with a suction device in the large slaughterhouse and
manually in the other one. Sampling was performed immediately
after spinal cord removal.
2.2. Alternative techniques
Two alternative spinal cord removal techniques (Figs. 1 and 2)
were compared to the conventional method. In both techniques,
the spinal cord is extracted after decapitation and before the
carcass is split. As shown in Fig. 1, the rst technique (LGR equip-
ment, Portile, Italy), already adopted by some Member States,
entails sucking the spinal cord out under vacuum through a PVC
hose previously inserted into the vertebral canal through the atlas
and cautiously advanced caudally. The other technique (Piscopo,
2001) involves removing the spinal cord by water-jet (Fig. 2). A
hose pipe (18 mm in diameter) connected to a tap is inserted into
the vertebral canal through the sacrum; the water-jet pressure (3e
4 bar) causes the spinal cord to leak out through the atlas.
The feasibility of these alternative innovative techniques in eld
conditions was evaluated by monitoring the average time each
technique took to complete and its overall complexity of applica-
tion during slaughtering.
2.3. Comparative study
The estimated sample size was 50 carcasses for each spinal cord
removal method: conventional; suction; and water-jet. The speci-
mens were withdrawn immediately after carcass splitting. In this
context, it is to be stressed that carcass washing is forbidden in Italy.
The comparative study was carried out in the large slaughterhouse.
2.4. Sample collection and preparation
Bovine older than 12 months were included in the study.
Samples were collected using sterile swabs (Copan Italia, Brescia,
Italy) from a dened area on the medial surface of each half of the
split carcass. A 10 10 cm area was selected and marked off on the
paravertebral muscles (Fig. 3). The swabs were then repeatedly
bathed in 1 ml of sample dilution buffer (Ridascreen, R-Biopharm,
Darmstadt, Germany) and disposed. The buffer samples were
stored at 4

C until analysis the next day.
2.5. Testing activity
The RIDASCREEN

Risk Material 10/5 is a sandwich enzyme

immunoassay for the quantitative analysis of risk material (CNS) in/
D. Pitardi et al. / Food Control 30 (2013) 668e674 669
on rawmeat, meat products and on contaminated surfaces. In order
to facilitate application of the test for screening purposes, in
previous studies we validated its qualitative use by plotting an ROC
curve to set a useful cut-off value.
A volume of 50 ml of standards (0, 0.1, 0.2 and 0.4% CNS
concentration) in duplicate and 50 ml of samples were distributed
into wells originally coated with the anti-GFAP primary antibody.
Next, 50 ml of conjugate were added to each well, and the test plate
was incubated at room temperature for 10 min. After three-step
washing, 100 ml of substrate/chromogen were added prior to
incubation 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 for
each well. The RIDA SOFT Win software (R-Biopharm AG) allows
automated data retrieval from the reader and calculation of the
results by linear regression. In case of positive results, samples were
immediately retested in duplicate.
3. Statistics
The sample size for determining the prevalence level for
contamination at the slaughterhouse was calculated by taking into
account the results of the previous study by the same work group
(Bozzetta et al., 2006). A prevalence of contamination of 15%, a 95%
condence level, and an accepted error of 4% were assumed to
estimate the sample as representative of the two abattoirs.
The sample size necessary to evaluate differences among the
three removal methods was obtained by tting the Kastenbaum,
Hoel and Bowman tables to our data Kastenbaum, Hoel e Bowman
tables 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 channel
with caution, the spinal cord is then extracted under vacuum.
D. Pitardi et al. / Food Control 30 (2013) 668e674 670
evaluated by means of a test for normality based on skewness and
kurtosis. As the data obtained were not normally distributed, the
KruskaleWallis equality-of-populations rank test was performed to
verify the key differences between contamination levels associated
with the three SRMremoval techniques. The data were entered into
an ad hoc database and analysed using Stata 11 SE software.
4. Results
Intherst stage of this researchwecarriedout a surveyinorder to
evaluatethelevel of CNTcontaminationusingconventional slaughter
technique. A total of 412 bovine carcasses were investigated after
splitting and spinal cord removal in two slaughterhouses; one large
andtheother medium-sized. Samples were denedas positive if CNS
tissue was detected at a concentration 0.049%.
The lowest level of CNT contamination was detected in the
large slaughterhouse applying the suction technique; samples
tested positive in 130 out of 216 carcasses (60.2%, 95% CI [con-
dence interval], 53.3e66.8). In the medium-sized one, the spinal
cord was manually removed and 152 out of 196 carcasses tested
positive (77.6%, 95% CI, 71e83.2). Table 1 summarizes the results
stratied by contamination level. Lastly, the survey data assessed
were used to establish the overall prevalence of CNT contamina-
tion connected with the conventional slaughter practice: an
Fig. 2. (continued).
D. Pitardi et al. / Food Control 30 (2013) 668e674 671
overall prevalence of CNT contamination of 68.4% (95% CI, 53e83)
was shown.
In the second stage of this study we compared the conventional
practice with two alternative methods by which the SRM is
extracted before the carcass is split. The sample size was 50
carcasses for each SRM removal method.
The comparative study showed a CNS contamination of 62%
(95% CI, 47.2e75.3) associated with the conventional technique,
60% (95% CI, 45.2e73.6) with the suction technique, and 36% (95%
CI, 22.9e50.8) with the water-jet system (Fig. 4). The difference
among the three methods appeared to be signicant (P 0.0047)
(Table 2). The percentages of contamination stratied according to
the 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 in
conventional 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; Kale
et al., 2008; Lim et al., 2007; Love et al., 2000; Prendergast et al.,
2003; Takada et al., 2008). Carcass splitting has been identied as
the key stage for contamination by SRM, due to the mixture of
sawing residues and rinsing water, or sawing sludge, that collects
in 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 which
the SRM is extracted prior to splitting (Piscopo, 2001; Ramantanis,
2006; Troeger, 2004).
The aim of our study was to estimate the prevalence of SRM
contamination of carcasses on the medial split surface of the
paravertebral 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 two
alternative techniques in which the carcass is split after spinal
cord removal.
Table 1
Conventional method epositive samples stratied according to contamination level
and type of abattoir.
CNS contamination Large abattoir
no. positive
samples (%)
Medium abattoir
no. positive
samples (%)
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%
Traditional Suction Water-jet
Negative samples Positive samples
Fig. 4. The histogram shows the percentages of positive and negative samples grouped
by method.
Fig. 3. Schematic diagram of the area swabbed on the medial surface of the split
carcass.
D. Pitardi et al. / Food Control 30 (2013) 668e674 672
The ELISA RRM 10/5 was applied as a qualitative assay. The
estimated cut-off value excludes a possible reactivity related to the
peripheral nerve counterpart (Bozzetta et al., 2006).
In previous studies, assessment of the contamination levels in
different sampling areas of the carcass indicated that CNS
contamination is greater on the internal surfaces along the cut
vertebral surface, where the lumbar and cervical regions were
found to be the most contaminated (Helps et al., 2002; Prendergast
et al., 2003). Accordingly, we selected as the target sampling area
the paravertebral muscles of the cervical region.
The highest percentage of CNS contamination was associated
with the conventional slaughtering method. When the positive
samples were stratied 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 handling
techniques or the suction device used to eliminate the spinal cord
after carcass splitting, which might require less carcass manipula-
tion than manual removal as employed in the medium-sized
facility.
When compared against the conventional method, the two
alternative slaughter techniques were associated with a lower level
of CNS contamination on paravertebral meat. However, contami-
nation after spinal cord removal by suction was only slightly less
than the one obtained using the traditional method (60 vs. 62%,
respectively). This may be explained by the fact that the spinal 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 the
vertebral channel, which may not always be realistic under eld
conditions. Other problems were related to the frequent hose
clogging with spinal cord tissue and the occurrence of vertebral
bone breaks or dislocations.
On the other hand, the spinal cord removal with the water-jet
was associated with a 30% reduction in CNS contamination: 89%
of positive samples had a low level of CNS contamination. The
technique is quick and easy to be applied, it doesnt require any
dedicated training. When the carcass is located in the splitting area
the sacrum is cut along its length to display the vertebral channel;
a hose pipe connected to a water tap is inserted into the vertebral
channel 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 cord
from each carcass and the results showed that the removal of the
whole spinal cord before splitting prevents the spread of the
contamination on the carcass surface.
The level of CNS contamination still present on carcasses treated
by this method was further investigated. Since previous studies
demonstrated that the splitting saw plays an important role in
disseminating the CNS tissue from one carcass to the next (Helps
et al., 2004), the rst and the last-slaughtered carcasses were day
by day sampled and analysed after applying the water jet treat-
ment, as to monitor the spread of the contamination. Under such
simulated abattoir conditions, the remaining contamination rate
was found out to be effectively related to the scatter of the CNS
tissue remaining in the saw housing from one carcass to another
(data not shown). Difculties resulting from occasional breaks or
dislocation of vertebral bones have also been reported with this
method.
6. Conclusions
According to EC Regulation 999/2001, specied risk materials
and possibly CNS tissues have been banned from entering the
human food chain, as these tissues harbour the highest level of
infection. Nevertheless, CNS tissues can still contaminate carcasses
during slaughtering. This could mean that if an animal with sub-
clinical BSE or an undiagnosed infected animal is slaughtered, the
infectious agent (PrPsc) could spread over the carcass, potentially
exposing 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 justied by the wide use of highly sensitive
and specic BSE rapid tests for screening purposes.
In light of the encouraging trends in the control of BSE, the
European Commission has outlined possible future changes to EU
measures on TSEs, introducing a gradual risk-based lift of the feed
ban and an increase of the age limit for testing (European
Commission, Brussels, 16.7.2010). In this context, the develop-
ment of alternative strategies, and their application to slaughter
methods, could inform risk-based policies. This study incontro-
vertibly shows that an alternative slaughter practice can reduce the
CNS contamination of meat products and help to protect
consumers against the risk of exposure to BSE.
Acknowledgements
This study was funded by the Italian Ministry of Health (Project
IZSPLV 03/08RC).
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