Content uploaded by Miyako Yoshioka
Author content
All content in this area was uploaded by Miyako Yoshioka on Jul 15, 2014
Content may be subject to copyright.
Available via license: CC BY 2.0
Content may be subject to copyright.
RES E AR C H A R T I C L E Open Access
Rapid assessment of bovine spongiform
encephalopathy prion inactivation by heat
treatment in yellow grease produced in the
industrial manufacturing process of meat
and bone meals
Miyako Yoshioka
1,2
, Yuichi Matsuura
1
, Hiroyuki Okada
1
, Noriko Shimozaki
1
, Tomoaki Yamamura
1
,
Yuichi Murayama
1*
, Takashi Yokoyama
1
and Shirou Mohri
1
Abstract
Background: Prions, infectious agents associated with transmissible spongiform encephalopathy, are primarily
composed of the misfolded and pathogenic form (PrP
Sc
) of the host-encoded prion protein. Because PrP
Sc
retains
infectivity after undergoing routine sterilizing processes, the cause of bovine spongiform encephalopathy (BSE)
outbreaks are suspected to be feeding cattle meat and bon e meals (MBMs) contaminated with the prion. To assess
the validity of prion inactivation by heat treatment in yellow grease, which is produced in the industrial
manufacturing process of MBMs, we pooled, homogenized, and heat treated the spinal cords of BSE-infected cows
under various experimental conditions.
Results: Prion inactivation was analyzed quantitatively in terms of the infectivity and PrP
Sc
of the treated samples.
Following treatment at 140°C for 1 h, infe ctivity was reduced to 1/35 of that of the untreated samples. Treatment at
180°C for 3 h was required to reduce infectivity. However, PrP
Sc
was detected in all heat-treated samples by using
the protein misfolding cyclic amplification (PMCA) technique, which amplifies PrP
Sc
in vitro. Quantitative analysis of
the inactivation efficiency of BSE PrP
Sc
was possible with the introduction of the PMCA
50
, which is the dilution ratio
of 10% homogenate needed to yield 50% positivity for PrP
Sc
in amplified samples.
Conclusions: Log PMCA
50
exhibited a strong linear correlation with the transmission rate in the bioassay; infectivity
was no longer detected when the log PMCA
50
of the inoculated sample was reduced to 1.75. The quantitative
PMCA assay may be useful for safety evaluation for recycling and effective utilization of MBMs as an organic
resource.
Keywords: Prion inactivation, Bovine spongiform encephalopathy, Meat and bone meal, Yellow grease, Infectivity,
Protein misfolding cyclic amplification
* Correspondence: ymura@affrc.go.jp
1
Prion Disease Research Center, National Institute of Animal Health, 3-1-5
Kannondai, Tsukuba, Ibaraki 305-0856, Japan
Full list of author information is available at the end of the article
© 2013 Yoshioka et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Yoshioka et al. BMC Veterinary Research 2013, 9:134
http://www.biomedcentral.com/1746-6148/9/134
Background
Transmissible spongiform encephalopathies (TSEs), in-
cluding scrapie in sheep and goats, chronic wasting disease
(CWD) in deer and elk, bovine spongiform encephalopathy
(BSE) in cattle, and Creutzfeldt–Jakob disease (CJD) in
humans, are infectious and fatal neurodegenerative dis-
eases [1]. Proteinaceous infectious agents called prions are
thought to be responsible for TSEs, which are character-
ized by the accumulation of the pathogenic form of prion
protein (PrP
Sc
) in the nervous tissues of infected subjects
[2,3]. PrP
Sc
is a conformational isoform of the normal
cellular prion protein (PrP
C
), which is rich in beta-sheet
structures, insoluble in mild detergents, and resistant to
protease digestion [4,5].
Because prions retain infectivity after undergoing rou-
tine sterilization processes [6], contaminated meat and
bone meals (MBMs) are suspected to be the source of
BSE infection [7,8]. MBMs are manufactured through a
multi-step process involving the crushing of carcasses in
a pre-breaker, heating at 120°C–140°C in yellow grease
(lower-quality grades of tallow) in a cooker, and degreas-
ing from solid material by an oil separator. To determine
BSE prion inactivation during the manufacturing process
of MBMs, industrial proce sses were replicated on a pilot
scale by using BSE-infe cted brains, and the infectivity of
processed materials in each step was investigated in de-
tail [9]. However, in reality, processing conditions for
MBMs differ among rendering houses producing com-
mercial MBMs. Since the efficiency of prion ina ctivation
could be influenced by various factors such as treatment
temperature, time, steam pressure in the cooker, size,
water and fat contents of carcasses [10-13], it is difficult
to identify the risks attributable to specific processing
conditions. Furthermore, PrP
Sc
retained in the manufac-
turing process of MBMs remains to be elucidated.
The governments of many countries prohibited the
feeding of bovine MBMs following the feeding ban on
MBMs in the United Kingdom. A prion detection
method with high sensitivity and high accuracy must be
developed so that MBMs can be used safely in the
future. In addition, BSE prion is more resistant to phys-
ical and chemical treatment s than are scrapie and CJD
prions [14]. Therefore, experiments using BSE-infected
materials are essential for the assessment of BSE prion
inactivation as they can be considered a worst case among
prions. In recent years, it has become possible to perform
in vitro amplification of PrP
Sc
derived from various ani-
mals [15-21] by using protein misfolding cyclic amplifica-
tion (PMCA) [22]. We developed an ultrasensitive method
for BSE PrP
Sc
detection using potassium dextran sulfate
(DSP) [20]. The PMCA technique can also be used to
quantitatively assess scrapie PrP
Sc
[23-25], and our PMCA
method can be applied as an effective test for the assess-
ment of prion inactivation by monitoring residual BSE
PrP
Sc
[26]. In the present study, we investigated efficiency
of BSE prion inactivation following heat treatment in yel-
low grease by bioassay and quantitative PMCA.
Results
Infectivity of heat-treated homogenates
Long-term follow-up confirmed infectivity in the mice
intracerebrally inoculated with up to a 10
–5
dilution of
the 10% homogenate of the pooled spinal cords (Table 1).
PrP
Sc
accumulation was confirmed in the brains of the
diseased mice by western blotting and histopathological
analysis (data not shown). The infectious titer of the
homogenate was estimated to be 10
6.7
LD
50
per gram. A
strong linear correlation (r = 0.99) between the incuba-
tion times and dilution ratios of the inoculated hom-
ogenate was observed in mice inoculated with up to a
10
–3
dilution. Some mice inoculated with 10
–4
and 10
–5
diluted samples developed the disease after similar
prolonged survival times (735 or 736 days). In the ex-
treme dilution range, lower rate of transmission and
prolonged incubation time are generally observed in the
mice intracerebrally inoculated with prion-infected brain
homogenates. Since PrP
Sc
tends to aggregate, these phe-
nomena may be due to the near-absence of PrP
Sc
which
would have been almost completely diluted out.
Table 2 shows the effect of various heat treatments in
yellow grease on the BSE-infected spinal cord homoge-
nates. All mice inoculated with samples treated at 140°C
for 1 h died after an average of 304 days. The infectivity
was reduced to approximately 1/35 (log reduction = 1.54)
following the heat treatment. When the samples subjected
to temperatures above 140°C were used, 100% (180°C for
1 h) and 67% (160°C for 1 h) of the mice developed the
disease after prolonged average survival times. Regarding
the treatments for 3 h, infectivity was still detected in
some mice inoculated with the samples treated at 140°C
or 160°C. Because the incubation times of these diseased
mice were beyond the range of application of the regres-
sion line obtained using the titrated BSE-infected homoge-
nates, the log reduction of infectivity in each sample was
estimated to be more than 3.0. Meanwhile, mice inocu-
lated with samples treated at 180°C for 3 h did not exhibit
disease onset 790 days after inoculation.
PrP
Sc
detection by PMCA
Figure 1a illustrates the results of the amplification
of the samples subjected to the grease-heating method.
No PrP
Sc
signals were detected in the heat-treated sam-
ples by western blotting before amplification (data not
shown). After one round of amplification, PrP
Sc
signals
were dete cted in the samples treated at 140°C–180°C for
1 h and at 140°C for 3 h. PrP
Sc
signals were also detected
in both duplicate samples treated at 160°C and 180°C for
3 h after two or three rounds of amplification. In the
Yoshioka et al. BMC Veterinary Research 2013, 9:134 Page 2 of 8
http://www.biomedcentral.com/1746-6148/9/134
samples treated at 180°C for 3 h, trace amounts of PrP
Sc
remained after the treatment, although infectivity was
not detected in the bioa ssay.
Quantitative analysis of PrP
Sc
Figure 1b shows the results of the amplification of each di-
luted sample of untreated BSE-infected spinal cord hom-
ogenate. PrP
Sc
present in 10
–7
dilution of the infected
homogenate was detected in all tubes after three rounds
of amplification. PrP
Sc
signals were detected in three of
the 10
–8
and one of the 10
–9
dilutions after three rounds
of amplification. However, no additional tubes became
positive for PrP
Sc
in these dilutions after four rounds of
amplification. No signals were detected in the more ex-
treme dilution ranges even after four rounds of amplifica-
tion. Thus, the PMCA
50
of the 10% homogenate was
calculated to be 10
8.5
units on the basis of the results
obtained from the fourth round of amplification.
To evaluate the PrP
Sc
inactivation efficiency of each heat
treatment, we estimated the PMCA
50
from the results
obtained at the fourth round of amplification of serial 10-
fold dilutions of heat-treated samples. Serial PMCA was
sufficiently sensitive to detect PrP
Sc
in these diluted sam-
ples (Figure 2). The log reduction of PMCA
50
values of
the heat-treated samples are shown in Table 2. Regarding
the treatments for 1 h, PrP
Sc
inactivation appeared to be
most efficient in the samples treated at 160°C. This finding
is concordant with the observations of partial transmission
of infectivity (67%) in the mice inoculated with this sample
and prolonged incubation times of the diseased mice. Log
PMCA
50
decreased with extended heat treatment time:
although 180°C for 3 h was the most effective treatment, it
was unable to completely inactivate the proportion of
PrP
Sc
that is amplifiable by serial PMCA.
Figure 3 shows the relationships between the transmis-
sion rate in the bioassay and the log PMCA
50
values of
the inoculated samples. A strong linear correlation (r =
0.97) wa s observed between the log PMCA
50
values and
transmission rate. When the log PMCA
50
exceeded 5.25,
the transmission rate in the bioassay reached 100% as
observed in the mice inoculated with samples treated at
140°C or 180°h for 1 h. Infectivity was not detected in
the mice when the log PMCA
50
of the inoculated sample
was reduced to 1.75.
Discussion
In this study, BSE prion inactivation was analyzed quan-
titatively in terms of infectivity and the PrP
Sc
contents of
the samples after heat treatment in yellow grea se.
Following treatment at 140°C for 1 h, which is the heat
treatment condition generally used for carcasses in ren-
dering houses in Japan, the infectivity of the BSE-
infected spinal cord homogenate was reduced to at most
1/35 of that of the untreated control samples; further-
more, PrP
Sc
retained its capability for in vitro propagation.
Because carcasses are usually heat treated in closed
cookers, prions are affected by the steam pressure from
the water contained in the carcass. If a sufficient amount
of water is present in the carcass, prion inactivation may
proceed more efficiently in the cooker than under atmos-
pheric pressure. However, some degree of BSE infectivity
was still detected after autoclaving at 133°C in spiked raw
materials with high infectivity levels [26]. Furthermore,
the precise effects of high-pressure steam on carcasses
submerged in yellow grease are not known. Therefore,
high-risk materials such as brains and spinal cord should
be excluded from the rendering process for effective in-
activation of BSE prion.
Table 1 Mean incubation time of TgBoPrP mice following
intracerebral inoculation of titrated bovine spongiform
encephalopathy (BSE)-infected spinal cord homogenate
10% Homogenate
dilution
Transmission rate
(diseased/total)
Mean incubation
time ± SD (days)
10
0
100% (7/7) 242 ± 14
10
–1
100% (7/7) 279 ± 12
10
–2
100% (5/5) 322 ± 42
10
–3
100% (6/6) 367 ± 53
10
–4
33% (2/6) 736, 736, >790
10
–5
17% (1/6) 735, >790
10
–6
0% (0/6) >790
Table 2 Effects of various heat treatments in yellow grease on BSE-infected spinal cord homogenates
Temperature Time (h) Transmission rate
(diseased/total)
Mean incubation
time ± SD (days)
Log reduction
of infectivity
Log reduction
of PMCA
50
140°C 1 100% (6/6) 304 ± 13
*,‡
1.54 2.75
3 83% (5/6) 382 ± 64, >790 >3.0 4.0
160°C 1 67% (4/6) 471 ± 80
*,†
, >790 >3.0 3.5
3 17% (1/6) 514, >790 >3.0 6.0
180°C 1 100% (6/6) 380 ± 25
†,‡
>3.0 3.25
3 0% (0/6) >790 >3.0 6.75
*,†,‡
Significant differences (*: p < 0.01; †, ‡: p < 0.05) were observed among mice with respect to the mean incubation times as indicated by identical
superscript character.
Yoshioka et al. BMC Veterinary Research 2013, 9:134 Page 3 of 8
http://www.biomedcentral.com/1746-6148/9/134
We previously examined residual infectivity and PrP
Sc
after heat treatment of scrapie-infected hamster brains
under various experimental conditions [27]. The PMCA
results were concordant with bioassay results. However,
BSE PrP
Sc
was detected in the samples treated at 180°C
for 3 h, although infectivity was not detected in the bio-
assay. There are several possible explanations for this dis-
crepancy between infectivity and PrP
Sc
occurrence. For
example, BSE PrP
Sc
might contain various forms of PrP
Sc
with different amplification properties and infectivity, and
a PMCA-compatible form of PrP
Sc
with low or no
infectivity might predominate after heat treatment and be
maintained over other forms throughout the amplification
process. However, in the present study, the log PMCA
50
values were strongly correlated with the transmission rate
in the bioassay (Figure 3), suggesting that such PMCA-
compatible but less-infectious PrP
Sc
was not selectively
amplified in vitro.
In our pre vious paper, we demonstrated our amplifica-
tion system was highly sensitive and accurate, and no
spontaneous generation of PrP
Sc
was observed in the
amplification of various kind of samples derived from
Figure 1 Detection of bovine spongiform encephalopathy (BSE) PrP
Sc
by serial potassium dextran sulfate-protein misfolding cyclic
amplification. (A) Homogenates (10%) of BSE-infected spinal cords treated in yellow grease at 140°C–180°C for 1 or 3 h were diluted 10
–1
with
the PrP
C
substrate and amplified by serial PMCA. Duplicate samples were analyzed after each round (R1–R4) of amplification by western blotting
after digestion with proteinase K. The lanes labeled “N” are samples in which only the PrP
C
substrate was treated in the same manner. Horizontal
lines indicate the positions of molecular-weight markers corresponding to 37, 25, 20, and 15 kDa. (B) Homogenates (10%) of the heat-untreated
BSE-infected spinal cords were diluted 10
–7
to 10
–10
with the PrP
C
substrate and amplified in four tubes by serial PMCA.
Figure 2 Quantitative analysis of bovine spongiform encephalopathy (BSE) PrP
Sc
in heat-treated samples. Homogenates (10%) of
BSE-infected spinal cords treated in yellow grease at 140–180°C for 1 or 3 h were diluted with the PrP
C
substrate and amplified by serial dextran
sulfate-protein misfolding cyclic amplification. The dilution ratios examined in each sample are indicated. Quadruplicate samples were analyzed
after each round (R1–R4) of amplification by western blotting after digestion with proteinase K. The lanes labeled “N” are samples in which only
the PrP
C
substrate was treated in the same manner. Horizontal lines indicate the positions of molecular-weight markers corresponding to 37, 25,
20, and 15 kDa.
Yoshioka et al. BMC Veterinary Research 2013, 9:134 Page 4 of 8
http://www.biomedcentral.com/1746-6148/9/134
uninfected animals [20]. Determination of PMCA
50
based on quadruplicate amplification was also done in
our previous study [26], and we confirmed that similar
PMCA
50
values (around 10
11
per gram) were obtained in
two independent studies. In the present study, the
PMCA
50
of the BSE-infected spinal cords was estimated
to be 10
11.6
per gram, which is approximately 80,000-
fold greater than the corresponding intracerebral LD
50
per gram (10
6.7
) determined by the bioassay. The
PMCA
50
/LD
50
per gram of BSE prion was considerably
higher than those of scrapie prion strains (160–4000
fold) [25]. If this ratio reflects the numbe r of PrP
Sc
parti-
cles that compose an infectious unit of prions, more
PrP
Sc
particles might participate in an infectious unit of
BSE prions; moreover, such a large mass of PrP
Sc
parti-
cles might be processed into several smaller ones with
lower infectivity in vivo.
Alternatively, PrP
Sc
accumulation might proceed in
animals inoculated with PrP
Sc
when the PrP
Sc
concen-
tration is below a specific cut-off, but the animals might
not develop the disease within their lifetimes. Actually,
clinically asymptomatic infections are known as the sub-
clinical infection stage [28-30]. In the present study,
we examined PrP
Sc
in brains of asymptomatic mice inoc-
ulated with titrated BSE-inf ected homogenate, and PrP
Sc
was found at various levels in four of five mice ino-
culated with 10
–6
dilution of the infected homogenate
(Figure 4). Therefore, pathogenicity might be detected
by serial transmission in anim als as in the case of serial
PMCA. If so, the detection sensitivity of the bioassay
used in the present stud y may not be sufficiently high
for proper safety evaluation, because ecycling of BSE-
infected bovine tissues possibly augments the concen-
tration of PrP
Sc
in commercial MBMs if the carcasses
contain infinitesimal amounts of prion.
Another aspect of heat treatment in yellow grease is
that higher-temperature treatments do not necessarily
inactivate BSE prion more effectively. In the case of heat
treatment for 1 h, the results of both the bioassay and
PMCA indicate that BSE prion inactivation proceeded
more effectively with treatment at 160°C rather than
at 180°C. Samples treated at 180°C were dark brown,
suggesting that the surface was scorched during treat-
ment. In such high-temperature conditions, thermal
conduction may be inhibited by scorching of the sample
periphery, consequently requiring longer treatment time
to reach thermal equilibrium in the sample. Extension of
the treatme nt time to 3 h was actually necessary for the
loss of infectivity. However, further studies are needed to
confirm the above possibility.
Conclusions
In this study, we demonstrated that heat treatment at
180°C for 3 h is required for the loss of infectivity of BSE
prion in grease heating in our experimental conditions.
Furthermore, BSE PrP
Sc
retains amplification ability even
after such a treatment. The inactivation efficiency of BSE
PrP
Sc
could be quantitatively analyzed with the introduc-
tion of the PMCA
50
, which is strongly correlated with the
transmission rate in the bioassay. The serial PMCA tech-
nique is more practical and less time consuming than bio-
assays, and may be applicable for monitoring residual
Figure 3 Relationship between the log PMCA
50
and
transmission rate in the bioassay. A strong linear relationship
(r = 0.97) was observed in the samples treated at 140°C–180°C for
1h(●) and 3 h (▲).
Figure 4 Detection of PrP
Sc
in brains of asymptomatic mice. A
10% brain homogenate from five (#1-#5) of six asymptomatic mice
inoculated with 10
–6
dilution of infected homogenate (Table 1) was
prepared, and amplified by serial PMCA. Quadruplicate samples were
analyzed after each round (R1–R4) of amplification by western
blotting after digestion with proteinase K. One (#1 and #3), two (#5)
and four (#2) of the quadruplicate samples were found to be
positive for PrP
Sc
after three or four rounds of amplification. No PrP
Sc
signal was detected in #4 mouse. The eight lanes labeled “N” are
samples in which only the PrP
C
substrate was treated in the same
manner. Horizontal lines indicate the positions of molecular-weight
markers corresponding to 37, 25, 20, and 15 kDa. nt: not tested.
Yoshioka et al. BMC Veterinary Research 2013, 9:134 Page 5 of 8
http://www.biomedcentral.com/1746-6148/9/134
PrP
Sc
in the other steps of the manufacturing of MBMs
and useful for safety evaluation for recycling and effective
utilization of MBMs as an organic resource.
Methods
Experimental heat treatment procedure
All animal experiments were approved by the Animal
Care and Use Committee of the National Institute of
Animal Health (approval IDs: 450 and 08-008) in ac-
cordance with the Guidelines for Animal Transmissible
Spongiform Encephalopathy Experiments of the Ministry
of Agriculture, Forestry, and Fisheries of Japan. Spinal
cords were obtained from four cows experimentally in-
oculated with BSE at the terminal stage of the disease.
The infected materials were pooled and homogenized
using a blender. Pure homogenate (0.5 g) was placed on
a strip of aluminum foil (2 cm × 2 cm) and stored at –
80°C until further use. For use, the homogenate with the
aluminum foil was thawed at room temperature and
then immersed in 15 mL yellow grease preheated to
140°C, 160°C, or 180°C in a ceramic crucible by using an
electric heating device (ND-M11, Nissin Rika, Tokyo,
Japan). The yellow grease used wa s obtained from a ren-
dering house in Japan. The crucible was covered, and a
thermosensor was inserted through a hole in the cover
to monitor the temperature of the yellow grease. The
yellow grease was, then, kept for 1 or 3 h at the desired
temperature. The homogenate sample firmly adhered to
the surface of aluminum foil and was not broken into
pieces during the heat treatment. After the treatment,
the homogenate with the aluminum foil was removed
from the yellow grease with tweezers and placed on a
paper towel for absorption of the excess yellow grease.
The weights of the homogenates were reduced to 60–
70% of their original weights. The resultant materials
were thoroughly crushed with a mortar, and suspended
in PBS at 10% (w/v). Insoluble materials were separated
by brief centrifugation, and aqueous fraction was stored
at –80°C until further use.
Bioassay
Infectivity titer using transgenic mice overexpressing bo-
vine PrP
C
is generally 100-1000 times higher than that
using cows. Therefore, more accurate estimation of BSE
infectivity is able to be conducted by using such mice.
The heat-treated samples were injected intracerebrally
into six Tg(BoPrP)4092HOZ/Prnp
0/0
(TgBoPrP) trans-
genic mice (20 μL per mouse) overexpressing bovine
PrP
C
[31]. To determine the infe ctivity titer, serial 10-
fold dilutions of the 10% homogenate of the untreated
spinal cords were prepared in PBS and injected intrace-
rebrally into five to seven TgBoPrP mice (20 μL per
mouse). After inoculation, the mice were evaluated daily
for signs of infection. The lethal dose (LD
50
)was
determined according to the 50% endpoint calculation
method. Mean incubation times of the diseased mice
were analyzed by one-way ANOVA and Tukey’s multiple
comparison test.
PMCA
Bovine PrP
Sc
was amplified as described previously [20].
Briefly, the brains of TgBoPrP transgenic mice and PrP
knockout (PrP
0/0
) m ice were homogenized separately in
PBS containing 1% Triton X-100 and 4 m mol L
–1
EDTA. After centrifugation at 4500 × g for 5 min, the
supernatants were mixed in PrP
0/0
/TgBoPrP (5:1). A
mixturecontaining0.5%DSPwasusedasthePrP
C
sub-
strate for PMC A.
The 10% homogenates of heat-treated samples were
mixed at 1:9 with the PrP
C
substrate (total volume,
100 μL) in electron beam-irradiated polystyrene tubes.
Amplification was performed in duplicate with a fully
automatic cross-ultrasonic protein-activating apparatus
(Elestein 070-CPR, Elekon Science, Chiba, Japan), which
has a capacity to generate high ultrasonic power (700 W).
PMCA amplification was performed by 40 cycles of sonic-
ation (3-s pulse oscillations repeated 5 times at 1-s inter-
vals), followed by incubation at 37°C for 1 h with agitation.
For serial PMCA, 1:5 dilution of the PMCA product and
subsequent amplification was repeated twice.
To evaluate the inactivation efficiency of BSE PrP
Sc
by
heat treatment, the PMCA
50
, which is the dilution ratio of
the 10% homogenate needed to yield 50% PrP
Sc
positivity
for amplified samples, was determined. Serial 10-fold dilu-
tions of the 10% homogenate of the heat-treated and un-
treated samples were prepared and mixed 1:9 with the
PrP
C
substrate (total volume, 80 μL) and amplified in elec-
tron beam-irradiated eight-strip polystyrene tubes (076-
96, Elekon Science). Amplification was performed in
quadruplicate using 40 cycles of sonication (pulse oscilla-
tion for 5 s, repeated 5 times at 1-s intervals), followed by
incubation at 37°C for 1 h with agitation. For serial
PMCA, 1:5 dilution of the amplified product and subse-
quent amplification was repeated 3 times. The PMCA
50
was estimated from the results of the fourth round of
amplification by using the 50% endpoint calculation
method.
Western blotting
The amplified samples (10 μL) were mixed with 10 μL
proteinase K solution (100 μgmL
–1
) and incubated at
37°C for 1 h. The digested samples were mixed with
20 μL 2× SDS sample buffer and incubated at 100°C for
5 min. The samples were separated by SDS-PAGE and
transferred onto a polyvinylidene fluoride membrane
(Millipore, Bedford, MA). After the membrane was
blocked, it was incubated for 30 min with a horseradish
peroxidase (HRP)-conjugated T2 monoclonal antibody
Yoshioka et al. BMC Veterinary Research 2013, 9:134 Page 6 of 8
http://www.biomedcentral.com/1746-6148/9/134
[32]. After washing, the blotted membrane was devel-
oped using the Luminata Forte Western HRP Substrate
(Millipore) according to the manufacturer’s instructions.
Chemiluminescence signals were analyzed with a Light
Capture System (Atto, Tokyo Japan).
Histopathological analysis
The left hemispheres of the brains were fixed in 10% buff-
ered formalin for neuropathological analysis. Coronal
brain sections were immersed in 98% formic acid to re-
duce infectivity and embedded in paraffin wax. Sections
(4 μm thick) were cut and stained with hematoxylin and
eosin, and analyzed immunehistochemically as described
previously [20].
Abbreviations
PrP
Sc
: Pathogenic form of prion protein; BSE: Bovine spongiform
encephalopathy; MBMs: Meat and bone meals; PMCA: Protein misfolding
cyclic amplification; TSEs: Transmissible spongiform encephalopathies;
CWD: Chronic wasting disease; CJD: Creutzfeldt–Jakob disease; PrP
C
: Normal
cellular prion protein.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MY and YM (Murayama) designed and prepared the manuscript. MY, YM
(Matsuura), HO and YM (Murayama) performed the experiments. NS and TY
helped to perform the experiments. TY and SM supervised the study. All
authors have read and approved the final manuscript.
Acknowledgments
We wish to thank the animal caretakers of the Prion Disease Research Center
of the National Institute of Animal Health for their assistance. This study was
funded by a grant from the Bovine Spongiform Encephalopathy Control
Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan.
Author details
1
Prion Disease Research Center, National Institute of Animal Health, 3-1-5
Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
2
Research Area of Pathology
and Pathophysiology, National Institute of Animal Health, 3-1-5 Kannondai,
Tsukuba, Ibaraki 305-0856, Japan.
Received: 25 March 2013 Accepted: 3 July 2013
Published: 9 July 2013
References
1. Collinge J: Prion diseases of humans and animals: their causes and
molecular basis. Annu Rev Neurosci 2001, 24:519–550.
2. Prusiner SB: Molecular biology of prion disease. Science 1991,
252(5012):1515–1522.
3. Prusiner SB: Prions. Proc Natl Acad Sci USA 1998, 95(23):13363–13383.
4. Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS: Secondary
structure analysis of the scrapie-associated protein PrP 27-30 in water by
infrared spectroscopy. Biochemistry 1991, 30(31):7672–7680.
5. Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I,
Huang Z, Fletterick RJ, Cohen FE, et al: Conversion of α-helics into
β-sheets features in the formation of the scrapie prion proteins. Proc Natl
Acad Sci USA 1993, 90(23):10962–10966.
6. Taylor DM: Resistance of transmissible spongiform encephalopathy
agents to decontamination. In Prions, A Challenge for Science, Medicine
and Public Health System. Edited by Rabenau HF, Ciantl J, Doerr HW. Basel:
Karger; 2001:58–67.
7. Wilesmith JW, Wells GA, Cranwell MP, Ryan JB: Bovine spongiform
encephalopathy: epidemiological studies. Vet Rec 1988, 123(25):638–644.
8. Butler D: Statistics suggest BSE now ‘Europe-wide’. Nature 1996, 382(6586):4.
9. Taylor DM: Inactivation of the bovine spongiform encephalopathy agent
by rendering procedures. Vet Rec 1995, 137(24):605–610.
10. Taylor DM: Inactivation of prions by physical and chemical means. J Hosp
Infect 1999, 43(Suppl 1):S69–S76.
11. Taylor DM, Fernie K, McConnell I, Steele PJ: Observations on thermostable
subpopulations of the unconventional agents that cause transmissible
degenerative encephalopathies.
Vet Microbiol
1998, 64(1):33–38.
12. Schreuder BEK, Geertsma RE, van Keulen LJ, van Asten JA, Enthoven P,
Oberthür RC, de Koeijer AA, Osterhaus AD: Studies on the efficacy of
hyperbaric rendering procedures in inactivation bovine spongiform
encephalopathy (BSE) and scrapie agents. Vet Rec 1998, 142(18):474–480.
13. Muller H, Stitz L, Wille H, Prusiner SB, Riesner D: Influence of water, fat,
glycerol on the mechanism of thermal prion inactivation. J Biol Chem
2007, 282(49):35855–35867.
14. Giles K, Glidden DV, Beckwith R, Seoanes R, Peretz D, DeArmond SJ,
Prusiner SB: Resistance of bovine spongiform encephalopathy (BSE)
prions to inactivation. PLoS Pathog 2008, 4(11):e1000206.
15. Saá P, Castilla J, Soto C: Ultra-efficient replication of infectious prions by
automated protein misfolding cyclic amplification. J Biol Chem 2006,
281(46):35245–35252.
16. Murayama Y, Yoshioka M, Yokoyama T, Iwamaru Y, Imamura M, Masujin K,
Yoshiba S, Mohri S: Efficient in vitro amplification of a mouse-adapted
scrapie prion protein. Neurosci Lett 2007, 413(3):270–273.
17. Kurt TD, Perrott MR, Wilusz CJ, Wilusz J, Supattapone S, Telling GC,
Zabel MD, Hoover EA: Efficient in vitro amplification of chronic wasting
disease PrP
RES
. J Virol 2007, 81(17):9605–9608.
18. Thorne L, Terry LA: In vitro amplification of PrP
Sc
derived from the brain
and blood of sheep infected with scrapie. J Gen Virol 2008,
89(12):3177–3184.
19. Jones M, Peden AH, Prowse CV, Gröner A, Manson JC, Turner ML, Ironside
JW, MacGregor IR, Head MW: In vitro amplification and detection of
variant Creutzfeldt-Jakob disease PrP
Sc
. J Pathol 2007, 213(1):21–26.
20. Murayama Y, Yoshioka M, Masujin K, Okada H, Iwamaru Y, Imamura M,
Matsuura Y, Fukuda S, Onoe S, Yokoyama T, et al: Sulfated dextrans
enhance in vitro amplification of bovine spongiform encephalopathy
PrP
Sc
and enable ultrasensitive detection of bovine PrP
Sc
. PLoS One 2010,
5(10):e13152.
21. Yokoyama T, Takeuchi A, Yamamoto M, Kitamoto T, Ironside JW, Morita M:
Heparin enhances the cell-protein misfolding cyclic amplification
efficiency of variant Creutzfeldt-Jakob disease. Neurosci Lett 2011,
498(2):119–123.
22. Saborio GP, Permanne B, Soto C: Sensitive detection of pathological prion
protein by cyclic amplification of protein misfolding. Nature 2001,
411(6839):810–813.
23. Chen B, Morales R, Barria MA, Soto C: Estimating prion concentration in
fluids and tissues by quantitative PMCA. Nat Meth 2010, 7(7):519–520.
24. Pritzkow S, Wagenführ K, Daus ML, Boerner S, Lemmer K, Thomzig A,
Mielke M, Beekes M: Quantitative detection and biological propagation of
scrapie seeding activity in vitro facilitate use of prions as model
pathogens for disinfection. PLoS One 2011, 6(5):e20384.
25. Makarava N, Savtchenko R, Alexeeva I, Rohwer RG, Baskakov IV: Fast and
ultrasensitive method for quantitating prion infectivity titre. Nat Commun
2012, 3:741.
26. Matsuura Y, Ishikawa Y, Bo X, Murayama Y, Yokoyama T, Somerville RA,
Kitamoto T, Mohri S: Quantitative analysis of wet-heat inactivation in
bovine spongiform encephalopathy. Biochem Biophys Res Commun 2013,
432(1):86–91.
27. Murayama Y, Yoshioka M, Horii H, Takata M, Yokoyama T, Sudo T, Sato K,
Shinagawa M, Mohri S: Protein misfolding cyclic amplification as a rapid
test for assessment of prion inactivation. Biochem Biophys Res Commun
2006, 348(2):758–762.
28. Race R, Chesebro B: Scrapie infectivity found in resistant species.
Nature 1998, 392(6678):770.
29. Hill AF, Joiner S, Linehan J, Desbruslais M, Lantos PL, Collinge J:
Species-barrier-independent prion replication in apparently resistant
species. Proc Natl Acad Sci USA 2000, 97(18):10248–10253.
30. Race R, Raines A, Raymond GJ, Caughey B, Chesebro B: Long-term
subclinical carrier state precedes scrapie replication and adaptation in a
resistant species: analogies to bovine spongiform encephalopathy and
variant Creutzfeldt-Jakob disease in humans. J Virol 2001,
75(21):10106–10112.
Yoshioka et al. BMC Veterinary Research 2013, 9:134 Page 7 of 8
http://www.biomedcentral.com/1746-6148/9/134
31. Scott MR, Safar J, Telling G, Nguyen O, Groth D, Torchia M, Koehler R, Tremblay
P, Walther D, Cohen FE, et al: Identification of a prion protein epitope
modulating transmission of bovine spongiform encephalopathy prions to
transgenic mice. Proc Natl Acad Sci USA 1997, 94(26):14279–14284.
32. Hayashi HK, Yokoyama T, Takata M, Iwamaru Y, Imamura M, Ushiki YK,
Shinagawa M: The N-terminal cleavage site of PrP
Sc
from BSE differs from
that of PrP
Sc
from scrapie. Biochem Biophys Res Commun 2005,
328(4):1024–1027.
doi:10.1186/1746-6148-9-134
Cite this article as: Yoshioka et al.: Rapid assessment of bovine
spongiform encephalopathy prion inactivation by heat treatment in
yellow grease produced in the industrial manufacturing process of
meat and bone meals. BMC Veterinary Research 2013 9:134.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Yoshioka et al. BMC Veterinary Research 2013, 9:134 Page 8 of 8
http://www.biomedcentral.com/1746-6148/9/134
