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SID 5
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Research Project Final Report
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SID 5 (Rev. 05/09)
Project identification
SE4006
Classical Swine Fever Virus survival in meat products
and diagnostic samples
Contractor
organisation(s)
Veterinary Laboratories Agency
Woodham Lane
New Haw
Surrey
KT15 3NB
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 14
£
234,062
start date ................
01 June 2005
end date .................
31/03/2010
6. It is Defra’s intention to publish this form.
Please confirm your agreement to do so. ................................................................................... YES
NO
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should be written in a clear and concise manner and represent a full account of the research project
which someone not closely associated with the project can follow.
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Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
Classical swine fever virus (CSFV) is a highly infectious disease of pigs that has important economic and
social implications for the pig production industry. Minimising losses caused by such diseases will become
increasingly important to ensure future food security. Identifying the importance that different factors have
on the likelihood of the introduction or spread of this exotic disease is important to allow proportionate
strategies, which mitigate the risk of disease introduction without imposing undue restrictions, to be
defined.
The objective of this project was to provide data that will help to reduce the level of uncertainty associated
with future assessments of the risks that products of porcine origin pose for introduction of disease.
Specifically we made use of CSFV infected material, generated as part of a parallel project investigating
CSFV vaccination, to provide additional information on the levels of virus that are likely to be present in
meat and other tissues from CSFV infected animals. We have also investigated the effect of temperate on
how long CSFV survives. Generation of data on the rate that the virus becomes inactivated in culture
medium at various key temperatures has allowed calculation of the temperature increase that is needed to
reduce the viral concentration by one log within the same time (this value is termed the Z value). For the
virus strain studied this value is 10C in tissue culture medium. This data can be use to estimate how long
virus may survive at temperatures other than those tested. This tissue culture medium data can be used
as a baseline to inform on the likely stability of the virus in other matrices, but further investigation of the
stability of the virus in meat tissues and products of porcine origin is still required.
We have also investigated the survival of the virus in serum samples incubated at 56C. Treatment of
samples at this temperature for 30 minutes is often used to “inactivate” viruses prior to processing of
serum samples outside of biosecure containment laboratories, for example for ELISA testing. Our data
indicate that treatment of CSFV positive sera for this length of time will only reduce the viral concentration
by around one log. Treatment of sera for longer periods at 56C impacted on the efficacy of a CSFV
antibody ELISA, indicating that procedures for handling CSFV infected, or potentially CSFV infected,
material should take into consideration both disease security and test efficacy issues.
In the event of a future outbreak of CSFV, emergency vaccination with live attenuated CSFV vaccines
could be considered to assist in reducing spread of the disease. One strategy could be a “vaccinate to kill”
or suppressive strategy where animals surrounding an infected premise are vaccinated with a live
attenuated vaccine to reduce the potential for the virus to spread. Current live attenuated vaccines do not
allow the identification of animals that are also infected by serological tests and so, in such a strategy, the
SID 5 (Rev. 05/09)
Page 2 of 14
vaccinated animals would be slaughtered to prevent them harbouring field virus undetected. It is uncertain
what risk the meat from such vaccinated animals would pose if it were to enter domestic food processes.
To address this issue we investigated how much challenge virus, as opposed to vaccine virus, is present
in certain tissues of animals that have been vaccinated but subsequently exposed to challenge virus prior
to the onset of full protective immunity. Challenge virus could be detected in the tonsils and lymph nodes
of some animals that were partially protected by the vaccination and thus would be difficult to identify, as
they would have fewer clinical signs. Further investigation of the level of challenge virus in muscle and
other tissues that are more likely to be present in meat and pork from these animals will be beneficial to
help estimate how much of a risk the meat from such animal pose.
A major factor that determines how much of a risk a potentially infected product represents is how much
virus a susceptible animal would need to ingest to cause infection. Previous data on the infectious dose of
classical swine fever, which is from intranasal infection experiments, indicate that as few as 10 TCID 50
units of the virus are required to cause infection. However, it is likely that a much higher level of virus will
be required to cause infection when material is eaten. We have therefore determined the amount of the
moderately virulent UK2000 virus that was required to cause infection when fed orally to pigs. Our results
indicate that a much higher level of this virus, namely 10 4.35 TCID50 (CI 10 3.64 - 10 5.27 TCID50), is required
to cause infection when fed to pigs. This indicates that the risk of a contaminated product to cause
infection may be much less than previously thought. However, this level of virus can be present in only a
few grams of tissue from an acutely infected animal. It is known that the virulence of a strain can affect
the dose required to cause infection and so further investigation of the oral dose of a more virulent strain
will be of benefit, to provide information on possible worst case scenarios.
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
Objective 1
To determine the thermal inactivation curves of classical swine fever virus (CSFV) in meat products
Objective 2
To investigate the viral loads of classical swine fever in tissues which are included in meat products.
Objective 3
To investigate the viral load in tissues used in meat products in vaccinated and challenged animals.
SID 5 (Rev. 05/09)
Page 3 of 14
Objective 4
To determine the oral ID50 of classical swine fever virus
Objective 5
To investigate the effect of temperature of diagnostic samples on virus and viral RNA survival. (Objective
removed)
Classical swine fever virus is a highly infectious disease of pigs that has substantial economic and social
implications for the pig production industry. Minimising losses caused by such diseases will become increasingly
important to ensure future food security. Identifying the importance that different factors have on the likelihood of
the introduction or spread of this exotic disease is required to allow proportionate strategies, which mitigate the
risk of disease introduction without imposing undue restrictions, to be defined. To estimate the risk that products
of porcine origin pose for the introduction of a disease such as classical swine fever it is necessary to know how
much of the agent is likely to be present in a product, how long it will survive in that product, what is the likelihood
that the product will contact a susceptible individual and how much of the product needs to be ingested by a
susceptible individual for it to cause infection. This project aimed to provide data to increase our knowledge of
some of these factors and thus reduce the uncertainties associated with future CSF risk assessments.
To investigate the viral loads of classical swine fever in tissues which are included in meat products.
This objective (2) aimed to add to the knowledge that already exists concerning the levels of virus found in tissues after
infection with classical swine fever virus. The levels found vary with the virulence of the infecting strain, age of animals and
stage of the disease (Farez & Morely). To provide definitive data on the viral load present under all these varying parameters
would require substantial investigations, using large numbers of animals infected with different strains, at different ages and
with samples harvested at different stages post infection. Such investigations were outside the scope of this project, rather our
aim was to exploit material generated as part of a separate project that involved experimental infections with recently isolated
strains of CSFV to add to the information that is already available. This will be useful to help refine estimates of the amount of
virus likely to be present in a product derived from an infected pig, and will indicate if these estimates are relevant to recently
isolated strains.
As part of project SE0778, we inoculated a number of 10-week-old domestic pigs with two moderately
virulent strains of CSFV; the UK2000 strain, that caused the last outbreak of CSF in the UK, and strain CBR/93, which was
isolated in Thailand in 1993 and is genetically diverse from many strains previously studied in this context.
We therefore measured the quantity of viable virus present in tissues from pigs infected with two strains of CSFV by
determining the TCID50 (Dose that causes infection of 50% of tissue cultures) per gram of tissue. These animals were either
infected by intranasal inoculation with 10 5 TCID50 of virus, or were infected by contact with inoculated pen mates. Samples
were harvested at post mortem, which was generally between 12 and 18 pays post inoculation, although in-contact infected
animals were usually euthanized at an earlier stage of infection. The highest viral loads were present in blood and blood rich
tissues, such as spleen and liver. Lower levels were present in tissues that comprise the principle components of pork and
pork products, namely skeletal muscle and fat. Levels in longissimus dorsi muscle (pork loin) samples ranged from detectable,
but below the level quantifiable by the assay ie <103 TCID50/gm, up to a maximum of 10
median value of muscle samples with quantifiable levels was 10
median level in fat samples was 10
3.6
TCID50 gram (IQR 10
0.8)
.
4
6.5
TCID50/gm in one animal. The
TCID50 per gram (Inter Quartile Range IQR 10
1.1).
The
We could not detected virus in skin samples, although this
result may have been affected by the difficulty in processing this sample type, as CSFV has been detected in skin biopsies
(Kaden et al 2007). Although direct comparisons with other data are complicated by differences in methodology of analysis,
and also in the way viral loads are reported (ie pfu/ml rather than TCID50 per gram), the values we have obtained are in a
similar range to those reported previously by a number of authors (summarised in Farez & Morely), indicating that the levels of
virus of these recent moderately virulent strains is similar to historic isolates, which are often more virulent. The one animal
SID 5 (Rev. 05/09)
Page 4 of 14
that we identified with 10
6.5
TCID50 per gram of muscle appears to have an unusually high level of virus in the muscle tissue
and could be considered to represent the worst-case senario. Our results, and those of others are much higher than were
reported by Mebus and colleagues (1993 and 1997). These authors reported a value of 10 1 pfu/gm in the muscle of production
age pigs. This discrepancy could be affected by the fact that a measure of pfu/ml (plaque forming unit per ml) does not
correspond directly to TCID50 measures and it should also be noted that the 10 1pfu/ml value is based on some assumptions as
many of the samples tested were below the level of detection. However, this lower value could be a more accurate reflection
of the viral load present in meat tissues as Mebus et al used production age pigs whereas our results, and those of other
authors that were in the similar range, were from animals that were much younger, and as such are more susceptible to acute
CSF infection, than when animals are slaughtered for pork production. Further investigations of the level of virus found in the
tissues of older animals of normal slaughter age would therefore be of benefit.
To determine the thermal inactivation curves of classical swine fever virus (CSFV) in meat products (amended to
focus on serum) (Objective 1)
It is also necessary to consider how long the virus will remain viable during any process or environmental condition to which
the product may be subjected. Porcine products are subjected to a wide range of treatments and processing, the aim of this
objective is to examine one parameter that affects virus stability, namely temperature. We therefore aimed to produce data on
the rate that CSFV becomes non-viable in tissues, particularly muscle, at different temperatures. To provide a baseline to
guide further experiments with valuable material from experimentally infected animals we generated data on the rate of
inactivation of CSFV in tissue culture media. Aliquots of the reference virus strain Alfort 187 were incubated at the key
temperatures of 65C, 60C, 56C, 35C and 25C.
These temperatures were selected to cover a range of conditions that
products containing virus may encounter. For example, EU rules (2002/99/EC) accepted that virus is inactivated at 70C
provided this temperature is reached throughout meat. 65C was selected as a temperature lower than this to examine how
long virus might survive if the target temperature for inactivation were not reached. 60C is of relevance for composting of
material that may contain waste meat products; 56C is often used to inactivate biological sample such a serum and thus has
relevance for biosecurity issues, 35C and 25 C were selected to give an indication of survival times at ambient temperatures.
TCID50 values were calculated for triplicate aliquots at 8 time points for each temperature. The inactivation experiments were
repeated 2 or 3 times for each temperatures and D values, or the time required for a 10 1 drop in the TCID50 value, were
calculated from linear regression analysis of values above the quantifiable limit of the assay (Table 1).
For example, the mean D-value at 65C was 1.64 minutes (SD 0.4), which indicates that a sample with a high viral load of say
106 TCID50/ml would have to be heated to 65C for 9.8 minutes to result in complete virus inactivation, whereas the D value at
25 C was 7.8 days, indicating that a sample with 106TCID50/ml would still contain viable virus up until around 46 days later.
Table 1 Mean D values for CSFV strain Alfort in tissue culture media
Temperature C
D value (SD)
65
1.64 min (0.43)
60
6.6 min (0.67)
56
171 min (77)
50
3.12 hour (0.79)
35
1.47 day (0.24)
25
7.8 day (3.9)
SID 5 (Rev. 05/09)
Page 5 of 14
Fig 1. Effect of temperature on the survival of classical swine fever virus.
Data points in blue are D-values obtained in tissue culture medium for the strain Alfort187. Values in pink are for
the strain UK2000 in infected serum.
Plotting the D values against temperature allows calculation of the Z value, or the temperature increase (C) required to
reduce the D value one log, which for this CSFV strain in tissue culture medium is 10C. So, assuming the data remains
linear outside of the range tested, estimations can be made about the virus survival at other temperatures. For example, it
would be predicted that at 15C it would require around 105 minutes or 70 days for a one-log drop in viral titre.
It was our original intension to determine the inactivation rate in tissue samples, particularly muscle tissues as it
forms the major component of pork. Unfortunately attempts at this were hampered due to the relatively low
starting level, and non-uniform distribution, of virus in this tissue. Although the level of virus is significant in terms
of being a risk of infection to pigs (see below), it is close to the level of detection of the tissue culture TCID 50
assay and increasing the concentration of muscle tissue applied to the cell cultures has a detrimental effect on
the cell monolayer. This caused technical difficulties in determining inactivation rates in meat tissue, which we are
addressing in new project SE4010. We therefore focused in this project on determining the rate of inactivation of
the virus in serum.
Serum samples of various origin are often routinely treated at 56C for 30 minutes to “inactivate” them prior to
handling outside of microbiological safety cabinets, for example for many ELISA tests.
Although 56C for 30
minutes may be sufficient to inactive some viruses, this temperature is generally only suitable for inactivation of
complement not viruses. The above results for inactivation of CSFV in tissue culture medium imply that it would
SID 5 (Rev. 05/09)
Page 6 of 14
require 177 minutes just to reduce the viral load by one log, indicating that 30 minutes may not be sufficient for
inactivation of Classical swine fever virus in serum (although the data at 56C in tissue culture medium are
somewhat at odds with those at other temperatures). Other reports (Aynaud et al) also indicate that 30min at
56C may inactivate some CSFV strains but not others. We therefore determined inactivation curves of CSFV in
serum samples obtained from animals inoculated with the UK2000 virus strain. Triplicate inactivation experiments
resulted in a mean D value for this virus of 38.3 minutes (SD 29min) (Data in pink on Fig 1). This result was
consistent with the survival data in tissue culture medium and clearly indicates that 30 minutes at 56C is not
sufficient to completely inactivate virus in serum with high viral titres.
Increasing the time of incubation at 56C to 90 minutes, although not sufficient to completely inactivate very high
titre sera would result in around a 2.3 log reduction in viable virus and could represent a compromise method to
apply to minimise the risk posed by sera for which there is little reason to suspect CSFV presence. We therefore
investigated the effect of incubating sera for 90 min at 56 C on detection of CSFV antibody using the PrioCheck
CSFV antibody ELISA. Compared to untreated sera, treating sera at 56C for 90 minutes reduced the percentage
inhibition values obtained in the ELISA for all samples. This had the result that some samples that would be
detected as inconclusive if not treated gave a negative result when treated, and some samples that were positive
when untreated became inconclusive when treated (Fig 2).
Affect of Heat Treatment on CSFV Ab ELISA
150
PI Heat inactivated
100
50
-50
50
100
150
Samples positive
without treatment
but inconclusive
with treatment
-50
PI Not heat inactivated
Samples inconslusive
without treatment but
negative with treatment
Fig 2 Heat treatment at 56C for 90 minutes reduced the efficacy of the CSFV Ab ELISA.
Dotted lines indicate the cut-off levels of the assay, Samples with a percentage inhibition (PI) >50% are
considered positive, PI< 30% are negative, PI 31%-50% are inconclusive.
These results indicate that procedures used for the treatment of samples that may potentially contain CSFV to
inactivate them prior to removal from containment facilities must appropriately consider disease security and also
the impact of such treatments on the validity of tests to be performed on such samples.
SID 5 (Rev. 05/09)
Page 7 of 14
Objective 3
To investigate the viral load in tissues used in meat products in vaccinated and challenged animals.
The aim of this part of the project was to investigate how much challenge virus may be present in animals that have been
vaccinated and subsequently exposed to challenge virus, to facilitate estimates of the risk that meat from animals, vaccinated
for example as part of a vaccinate to kill strategy, might pose. A “vaccinate to kill” or suppressive strategy would use
vaccination of animals around an infected premise, for example in the protection zone, to control spread of the followed by
slaughter of vaccinates to prevent them harbouring field virus undetected. It is uncertain what risk meat from such animals
would pose if it were to enter normal food production chains. Vaccinated herds would have to be closely monitored for the
absence of field virus, for example by the use of sentinel animals, and/or laboratory detection by PCR. However it is not
possible to screen every carcass and so the acceptance of the use of such meat requires more certain estimates of the risks
posed.
As part of project SE0778 we completed an experiment in which pigs were vaccinated with a highly effective live
attenuated vaccine, and two further experiments where vaccinated animals were challenged with different CSFV strains at
periods shortly after vaccination. The vaccine rapidly prevented spread of the challenge virus to animals in direct contact,
indicating that it would be effective in reducing spread of CSFV if used in a vaccinate to kill strategy. We analysed blood and
nasal swab samples of these animals with a quantitative PCR that detects all CSFV (ie vaccine and challenge strains). We
have not detected vaccine virus in the blood of animals that were only vaccinated, indicating that any virus detected using this
PCR assay on blood samples of vaccinated and challenged animals will likely be challenge virus. Some of the animals
challenged very early post vaccination (1 day) were not protected from challenge, developed clinical signs and had high levels
of CSFV RNA in blood. Other animals were partially protected by the vaccine and had reduced levels of viral RNA in the blood
(Fig3) and nasal swabs.
Fig 3: Total CSFV viral RNA loads in blood of animals vaccinated with attenuated vaccine and challenged 1 day later.
To investigate in more detail how much of the virus present was challenge virus, as opposed to the vaccine, we
examined tonsil and lymph node tissues of vaccinated animals subsequently exposed to challenge with
quantitative PCR assays that discriminate between the vaccine virus and the challenge strains used. We initially
developed assays, using locked nucleic acid probes, that specifically detect the challenge strains used in our
experimental studies but not the vaccine strain. We also developed an assay that specifically detected the
vaccine but not the challenge strains. This vaccine specific assay proved to be less sensitive than an alternative
assay, developed by Leifer et al, that also only detects the vaccine strain and so the Leifer assay was used
instead.
As it was anticipated it would be difficult to detect the vaccine virus in muscle due to its absence from blood, we
examined tissues in which CSFV usually accumulates at high levels (i.e. various lymph nodes and tonsils).
Vaccine RNA was detected in some of these tissues, with the tonsils having the highest level of vaccine RNA.
The vaccine strain could be detected in the tonsils of most of the animals vaccinated 5 days prior to challenge but
SID 5 (Rev. 05/09)
Page 8 of 14
was less prevalent in the tonsils of animals vaccinated at times closer to the challenge. Most animals that were
protected from clinical disease by vaccination at 5 days prior to challenge had no detectable RNA from the
challenge strain in the tonsil or lymph nodes, although one animal had a low level of the UK2000 virus RNA
present in tonsils. Animals that were not protected clinically by the vaccination had high levels of challenge virus
in these tissues. Notably, the UK2000 challenge strain could be detected in tonsils and lymph nodes of animals,
in the day –3 and –1 groups, that were either completely or partially protected clinically. This was not observed
with the CBR/93 strain (Table 2)
SID 5 (Rev. 05/09)
Page 9 of 14
Table 2
Viral RNA detected by PCR assays that differentiate vaccine from challenge strain in tonsil tissue from animals
vaccinated at various time points prior to challenge with A) the UK2000 strain and B) the CBR/93 strain
Total CSFV RNA
+/+/++
+
+/+
++++
++
+
++
+/++++
++++
++
++
+++
++++
++++
++++
++++
++++
++++
A
UK2000
Challenge
strain RNA
Nt
+/+
++++
+
+
++
+/+++
++++
++
+
++
+++
++++
Nt
Nt
++++
++++
Total CSFV RNA
+
+/+
+
+
+
+
+
+
+/+
++
++++
++++
+++
++
++++
++++
++++
B
CBR/93
Challenge
strain RNA
++
++++
nt
++++
++++
Group
Vaccinated
-5 dpc
Vaccinated
-3 dpc
Vaccinated
-1dpc
Unvaccinated
Pig ID
AD 2552
AD 2553
AD 2554
AD 2555
AD 2556
AD 2557
AD 2561
AD 2562
AD 2563
AD 2564
AD 2565
AD 2566
AD 2570
AD 2571
AD 2572
AD 2573
AD 2574
AD 2575
AD 2578
AD 2579
AD 2580
AD 2581
Group
Vaccinated
-5 dpc
Vaccinated
-3 dpc
Vaccinated
-1dpc
Unvaccinated
Pig ID
AE 3003
AE 3004
AE 3005
AE 3006
AE 3007
AE 3008
AE 3012
AE 3013
AE 3014
AE 3015
AE 3016
AE 3017
AE 3021
AE 3022
AE 3023
AE 3024
AE 3025
AE 3026
AE 3027
AE 3028
AE 3029
SID 5 (Rev. 05/09)
Page 10 of 14
Vaccine strain
RNA
Nt
+
++
+
++
+
+
+
+/+
+
Vaccine strain
RNA
+
+/+
+
+
+/+/+
+/+
+++
nt
-
Clinically protected,
low or no viral RNA
in blood
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Partially
Partially
Partially
No
No
No
No
No
No
Clinically protected,
low or no viral RNA
in blood
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Partially
No
No
Partially
Partially
Partially
No
No
No
+/- = inconclusive
+ = 103 to 104 viral genome copies/g RNA
++ = 105 to 106 viral genome copies/g RNA
+++ = 107 to 108 viral genome copies/g RNA
+++ = 109 or above viral genome copies/g RNA
nt = not tested
These data indicate that in the unlikely event that vaccinated animals are exposed to challenge virus very shortly
after vaccination, they may become infected and thus could harbour challenge virus. If this exposure is such that
the vaccine provides little protection, it would be possible to identify this infection reasonably easily by clinical
signs and molecular detection of the high levels of viral RNA in the blood. However, animals that are partially
protected would be more difficult to detect on clinical signs or by molecular detection as they have low levels of
virus in blood samples for short time periods. These data indicate that such animals can harbour challenge virus
in the tonsils and lymph nodes. The inclusion of sentinel animals would potentially facilitate identification of such
animals. Further analysis of archived muscle tissues from these animals would be beneficial to identify the level of
challenge virus present in regions of the carcass that are more likely to enter the food chain, and may help
answer questions on the level of risk represented by meat from such animals.
During the use of the vaccine specific PCR assay described by Leifer et al 2009, which has been applied to assist
detection of CSFV infection amongst vaccinated wild boar populations in France and Germany, we identified a
sequence difference in the primer binding sites in different vaccine preparations. During the use of this assay in
the field, discrepancies occurred between the PCR result and sequencing data: specifically sequencing had
demonstrated the presence of vaccine, whereas the vaccine-specific PCR had indicated the samples were not
vaccine. However, as these samples were positive with a PCR assay that detects all CSFV strains they were
initially considered to be CSFV positive animals rather than vaccinated animals. The identification of this
sequence difference in the primer lead to a collaborative study that has resulted in redesign of the vaccinespecific assay that detects all variants of the vaccine (Leifer et al 2010) and will hopefully improve the usefulness
of this assay to differentiate infection from vaccination.
Objective 4
To determine the oral ID50 of classical swine fever virus
An important input for estimating the risk of a particular activity to cause a disease incident following a pig
ingesting a product contaminated with CSFV, is knowledge on how much virus a susceptible animal has to eat to
become infected. Data previously used for estimating the infectious dose for CSFV indicated that a very low
dose, less than 10 TCID50, can result in disease. This is much lower than other viral hazards such as FMD and
SVS and previous estimates of the risk of meat products have highlighted this as a major source of uncertainty in
the models (Hartnett et al 2004). However, the 10 TCID50 value is based upon intranasal inoculation of very
young weaner pigs, with a highly virulent CSFV (Dahle and Liess 1995). It is likely that the dose required to cause
infection following ingestion will be higher than after intranasal inoculation. We therefore investigated what dose
of the moderately virulent strain UK2000 is required to cause disease when fed orally. Corn covered blister pack
baits, provided by IDT Biologika GmbH, were filled with ten-fold dilutions of media containing virus and fed to 5
groups of 6, ten-week old pigs. These blister packs are designed for the vaccination of wild boar and allowed
SID 5 (Rev. 05/09)
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introduction of the inoculum in a way that it is chewed, rather than directly swallowed. The rationale for this was
that if the entire inoculum were swallowed it would be inactivated by the low pH in the stomach, whereas the
method used allowed some contact of the inoculum with tissues in the mouth and tonsils, a primary site of virus
replication. It is hoped this is a closer representation of what might happen if an animal were to eat an infected
pork product than intranasal inoculation. Blood and nasal swab samples were every taken 2-3 days and virus
infection monitored by real time RT-PCR. The time that viral RNA was detected in the blood of individual animals,
compared with the time that virus was first detected in nasal swabs, allowed differentiation of animals infected by
the inoculum from those infected by contact with previously infected pen mates. Only animals fed the highest two
doses became infected (Table 3)
Dose fed (TCID50)
Number of pigs infected by
feeding
A
1.5x101
0/6
B
1.5x102
0/6
C
1.5x103
0/6
D
1.5x104
3/6
E
1.5x105
5/6
F
Intranasal inoculated controls
2/2
Group
Table 3: Infection of animals fed different doses of CSFV
From these data the oral infectious dose was calculated using a logistic regression model to be 10 4.35 TCID50 with
a 95% Confidence Interval of 10
3.64
to 10
5.27
TCID50. This suggests that the risk posed by pork products may be
less than was estimated by risk assessments that used lower values. However, the level of virus present in
muscle tissues still indicates that only a few grams of fresh infected tissue would need to be consumed to cause
an infection.
The virus used in this experiment is a recent genotype 2.1 isolate of moderate virulence and so can be
considered representative of strains that have caused recent outbreaks in Europe and elsewhere. It is known that
the virulence of a strain affects the dose required to infect animals intranasally, so it will be of benefit to determine
the oral infectious dose of a highly virulent strain so that information on a virus that could be considered likely to
occur and also the “worst case“ scenario are available.
Aynaud JM, et al., 1972 Peste porcine classique: les facteurs d'identification in vitro (marqueurs génétiques) du
virus en relation avec le pourvoir pathogène pour le porc. Ann. Rech. veter.
Dahle and Liess (1995). Comparative study with cloned classical swine fever virus strains ALFORT and GLENTORF; clinical
pathological, virological and serological findings in weaner pigs. Win.terarztl. Mschr
Farez, S. & Morley, R. S. (1997). Potential animal health hazards of pork and pork products. Rev Sci Tech 16,
65-78.
Hartnett., E., Adkin, A., Coburn, H., Hall, S., England, T., Marooney, C., Cooper, J., Cox, T., Miles, S., and
Wooldridge, M. VLA and SafetyCraft Ltd (2004). Risk assessment for the import of conatminated meat and
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meat products into Great Britain and the subsequent Exposure of GB livestock:. Edited by F. a. R. A. D.
Department of Environment: Department of Environment, Food and Rural Affairs (Defra) Publications, London
PB9527
Mebus, C. A., M. Pineda, J.M. et al (1997). Survival of several porcine viruses in different Spanish dry-cured
meat products Food chemistry 59, 555.
Mebus, C. A., House, C., Gonzalvo, F. R., Pineda, J. M., Tapiador, J., Pire, J. J., Bergada, J.,
Yedloutschnig, R. J., Sahu, S., Becerra, V. & Sanchez-Vizcaino, J. M. (1993). Survival of foot-and-mouth
disease, African swine fever, and hog cholera viruses in Spanish serrano cured hams and Iberian cured hams,
shoulders and loins. Food Microbiology 10, 133-143.
Leifer, I., Depner, K., Blome, S., Le Potier, M. F., Le Dimna, M., Beer, M. & Hoffmann, B. (2009).
Differentiation of C-strain "Riems" or CP7_E2alf vaccinated animals from animals infected by classical swine
fever virus field strains using real-time RT-PCR. J Virol Methods 158, 114-122.
References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
SID 5 (Rev. 05/09)
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Leifer, I., Everett, H., Hoffmann, B., Sosan, O., Crooke, H., Beer, M. & Blome, S. Escape of classical
swine fever C-strain vaccine virus from detection by C-strain specific real-time RT-PCR caused by a point
mutation in the primer-binding site. J Virol
ScienceDirect - Journal of Virological Methods : Escape of classical swine fever C-strain vaccine virus
from detection by C-strain specific real-time RT-PCR caused by a point mutation in the primer-binding site
Evaluation of a primer-probe energy transfer real-time PCR assay for detection of classical
swine fever virus
Xing-Juan Zhanga,b,1, Hongyan Xiac,d,1, Helen Everette, Olubukola Sosane, Helen Crookee, Sándor Belákc,d,
Frederik Widénd, Hua-Ji Qiua,*, Lihong Liud,* Manuscript in preparation
Poster presentation at 3rd Epizone Annual meeting
Everett, H., Sosan, O., and Crooke H. (2009b). A differential PCR approach to monitor Classical Swine
Fever Virus challenge strains during experimental infection of C-strain vaccinated pigs. In 3rd Epizone
Annual Meeting Antalya Turkey, 12-15th July
Poster presentation at Society for General Microbiology t
Everett, H., Sosan, O., and Crooke H. (2010). A differential PCR approach to monitor Classical Swine
Fever Virus challenge strains during experimental infection of C-strain vaccinated pigs. In SGM Spring
meeting 2010, Systems, Mechanisms and Microorganisms 29th March-1st April 2010
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