D12264.PDF

Rev. sci. tech. Off. int. Epiz., 2012, 31 (3), 761-775
A quantitative assessment of the risk
of introducing foot and mouth disease virus into
the United States via cloned bovine embryos
B. Asseged (1)*, B. Tameru (1), D. Nganwa (1), R. Fite (2) & T. Habtemariam (1)
(1) College of Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, AL 36088,
United States of America
(2) Animal and Plant Health Inspection Service (APHIS), United States Department of Agriculture (USDA),
4700 River Road, Riverdale, MD 20737, United States of America
*Corresponding author: [email protected]
Summary
The trade of livestock or their products between nations requires information on
the risk of introducing infectious agents such as foot and mouth disease virus
(FMDV). Although transmission pathways for FMDV vary, a recent concern in the
United States (USA) is that it might enter via cloned embryos. A quantitative risk
assessment model was developed to determine the scenarios (with
mathematical probabilities) that could lead to the introduction and maintenance
of FMDV via the importation of cloned bovine embryos. Using @RISK software
with Monte Carlo simulation involving 50,000 iterations, the probability of
introducing FMDV via cloned embryos was estimated to be 3.1 × 10–7. Given the
current cloning protocol, and assuming the annual importation of 250 to
1,700 (mean = 520) cloned embryos, the expected number of infected embryos
ranges from 1.1 × 10–7 to 4.4 × 10–3 (mean = 1.6 × 10–4) per year. Critical pathway
analysis showed that the risk of FMDV entering the USA by this route is
extremely low.
Keywords
Cloning – Critical pathway analysis – Foot and mouth disease – Monte Carlo simulation
– Quantitative risk assessment – United States.
Introduction
Cloning by somatic cell nuclear transfer (SCNT) is a
relatively new technique in which animals are produced
asexually by taking a differentiated somatic cell and fusing
it with, or microinjecting it into, an enucleated oocyte. The
donor cells are commonly obtained by biopsy from
selected adults (26, 41), of which there is an unlimited
supply (9). However, the cytoplast of a mature oocyte is
the only type of cell identified as having the capacity to
reprogramme the donor cell nucleus correctly to express
the genes required for early development (30).
Somatic cell nuclear transfer continues to be developed
and improved, but as yet there is no method that is
universally employed. The basic steps (Fig. 1) are common
to most SCNT procedures, but it is a complex, technically
demanding and inefficient process (10, 21, 27, 32).
Nevertheless, it is a promising technique for preserving and
propagating superior genes with known performance traits
(4, 10). This is of particular value in food animal
production because it may take years to prove the merit of
a sire or dam. Cloning by SCNT has been used successfully
to produce livestock of various species, including the
camel (40).
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B
A
Oocyte source
Nucleus source
Removal of nucleus
and polar body
from oocyte
D
C
Cultured somatic cells
(often fibroblast)
E
Cell injection
F
Electrofusion and activation
G
H
Embryo clone
I
Surrogate dam
Genitor
Clone (F0)
Sexual reproduction
I
Clone offspring (F1)
A: cell donor
B: oocyte donor
C: cultured cells
D: enucleation
E: cytoplast
F: injection of somatic cell into cytoplast
G: electrofusion of oocyte and cell membrane
H: embryo clone
Fig. 1
Main steps of somatic cell nuclear transfer
Source: European Food Safety Authority, 2008 (8)
I: embryo transfer into a surrogate dam generating
clone (F0); F1 generated by mating of the clone (F0)
with a normal partner
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Owing to the complexity of the technique (8) and its
variable outcomes (32), the focus of cloning has so far
centred on generating live animal clones from various
donor sources and culture conditions. A surviving dam
and live offspring are, therefore, indicators of successful
outcomes. As a result, most studies so far have
concentrated on the development and refinement of new
techniques and on the observation of developmental
abnormalities that occur in cloned offspring (13, 19, 21).
Studies directly addressing the possible risk of transfer of
infectious agents by cloning are relatively rare (11, 45).
The objective of this study was to assess the probability of
introduction of foot and mouth disease virus (FMDV) into
the United States (USA) via the importation of cloned
bovine embryos. Hopefully, it will enable regulatory
authorities to make informed decisions with regard to such
importations.
Materials and methods
Details of cloning technique
in relation to sanitary management
Animal cloning by SCNT (see Fig. 1) comprises five major
steps that can be grouped into two broad categories. They
include:
a) Selection, isolation and preparation of:
i) the donor cells
ii) the recipient cells.
b) The execution of:
iii)nuclear transfer
iv) embryo culture
v) transfer of embryos into recipients (8).
All of the steps involved in cloning have been described in
detail elsewhere (9, 10, 20, 21, 33, 40, 41). However, brief
accounts of each of them are provided here in an attempt
to pinpoint the potential steps at which specific disease
agents could gain access to the methodological pathway.
Given that our model examines the failure of mitigations
(along the cloning pathway) either to inactivate, remove, or
detect FMDV originating from the donor, storage was not
taken into account in the analysis.
Preparation of donor cells
Tissues are aseptically removed from the identified cell
donor, washed in phosphate buffered saline (PBS), minced
with surgical blades, and cultured in dishes containing
Dulbecco modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS). Once a confluent
fibroblast monolayer is obtained, the culture is passaged
with an enzymatic solution (0.25% trypsin and 0.05%
ethylenediamine tetraacetic acid [EDTA]). The
disaggregated and individual cells thus isolated are washed
with Hepes and used for immediate nuclear transfer (NT)
(9, 10).
Preparation of recipient cytoplast
Ovaries collected from commercially slaughtered females
are immersed in normal saline and transported to the
laboratory within 2–4 h. Cumulus–oocyte complexes
(C-O-Cs) are collected by aspirating the ovarian follicles
into Hepes-buffered medium 199 (H199). Selected
C-O-Cs are then washed twice in H199 and once in
bicarbonate-buffered medium (M199), and transferred
into in vitro maturation (IVM) medium (B199). After
18–20 h, the cumulus cells are removed by vortexing the
C-O-Cs in the presence of hyaluronidase, and they are
further washed in H199. The denuded oocytes are then
placed in the manipulation medium (Hepes-TCM-199)
(9, 10, 40). Manipulation involves physical removal of the
metaphase chromosomes and the polar body, using finely
controlled microsurgical instruments. Once these have
been removed, the remainder of the oocyte, surrounded by
zona pellucida (ZP), is termed a cytoplast (41).
Nuclear transfer and activation
of reconstructed embryos
Individual washed donor cells are transferred into the
perivitelline spaces of enucleated oocytes (the cytoplast)
with a micropipette. The couplets are then washed in
fusion medium (0.3 M mannitol, 50 µM CaCl2,
100 µM MgCl2, 500 µM Hepes) and fused by electrical
pulse. Reconstructed cells are activated 3–5 h after fusion,
using
a
combination
of
ionomycin
and
6-dimethylaminopurine in synthetic oviductal fluid (SOF)
for 3 h (4, 9, 41).
Embryo culture
Following three washes in SOF, couplets are cultured
individually for six to nine days in a chemically defined
medium (10, 41). After this time, embryos that have
developed successfully to morula/blastocyst stage are
selected and washed ten times by transfer through a series
of wells of fresh media. Washing with trypsin has similar
requirements in that embryos are washed five times, then
exposed to trypsin in a sixth and seventh wash and, finally,
washed five more times without trypsin. They are finally
observed under a microscope to ensure that the ZP is free
from extraneous debris (33). Embryos for export are
normally stored cryoprotected in glycerol or ethylene
glycol and kept frozen in liquid nitrogen (35).
Embryo transfer
Good quality embryos (now at a blastocyst stage) are
transferred into synchronised recipients, and carried to
term to produce live cloned offspring (21).
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various distribution functions (including Uniform, Pert,
and Beta) were used to calculate expected values at each
node using @Risk software (Version 5.7, Palisade
Corporation). The sum of the product of the probability
distributions P1–P4 and P5–P7, multiplied by P8–P10
(Table I), was considered to be the overall probability of
introducing FMDV into the USA via cloned embryos. In
the analyses, Monte Carlo simulations (a computerised
mathematical technique for iteratively evaluating a
deterministic model using sets of random values as inputs)
were set at 50,000 iterations. For any factor that has
inherent uncertainty, Monte Carlo simulation builds
models of possible outcomes by substituting values
randomly from a range of possible values (the probability
distribution). It then calculates the results repeatedly
(iterations), each time using a different set of random
Development of the scenario pathway
The authors applied epidemiological principles to develop
a structured knowledge base with regard to the possible
transfer of FMDV along the cloning pathway (see Fig. 1).
Using this knowledge base, a scenario tree (Fig. 2),
represented by a series of risk-associated events (for the
purpose of the risk assessment, these events are termed
‘nodes’) (Nodes 1 to 10), was developed. From this the
authors determined the conditions (with associated
statistical probabilities) that could lead to the introduction
and maintenance of FMDV in the cloning pathway. The
parameters for the occurrence of the FMDV infection event
at each node, represented by distribution functions to
capture variability, were estimated using published
literature (1, 2, 36). Based on the ranges of estimates,
INITIATING EVENT: Importation of cloned embryos
NODE 1
Is selected somatic cell donor infected?
P1
N
N
Can diagnostic tests fail to detect
FMDV-infected somatic cell donor?
P2
N
N
Y
Can the tissue culture remove FMDV from donor cells?
P3
N
Can diagnostic tests fail to detect
FMDV in tissue culture?
P4
Y
NODE 8
Y
Can the washing process remove
FMDV from the developing embryo?
P8
Y
N
O
N
NODE 4
Is the oocyte/C-O-C infected?
P6
NODE 6
Can the maturation and washing processes
remove FMDV from the oocyte?
P7
NODE 7
Y
Y
NODE 3
NODE 5
Y
Y
NODE 2
Is slaughtered oocyte donor infected?
P5
R
I
S
K
N
N
NODE 9
N
Can FMDV-infected embryos develop
into exportable blastocysts?
P9
Y
NODE 10
Y
Can FMDV infection be detected
in exportable blastocysts?
P10
N
Infected cloned embryos imported into the USA
C-O-C: cumulus–oocyte complex
FMDV: foot and mouth disease virus
Fig. 2
A scenario tree for the likelihood of introduction of foot and mouth disease virus into the USA via cloned embryos
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Table I
Summary of input parameters and their respective distributions
Node Description
Parameter/distribution
1
Infected somatic cell donor
RiskPert (15%, 20%, 50%)
2
Proportion of false negatives
RiskPert (1%, 3%, 30%)
3
Failure of somatic cell culture
RiskPert (0%, 4%, 5%)
treatment to remove FMDV from
infected cells
4
Failure of laboratory tests to
RiskPert (2%, 3%, 20%)
detect FMDV in cell culture
Total risk associated with somatic cell donor = P1*P2*P3*P4
5
Infected oocyte donor
RiskPert (15%, 20%, 50%)
6
Infected C-O-C
RiskUniform (0%, 27%)
7
Failure of oocyte cleansing/washing RiskUniform (0%, 5%)
Total risk associated with oocyte donors = P5*P6*P7
Total risk associated with source animals =
(P1*P2*P3*P4) + (P5*P6*P7)
8
Failure of embryo washing
RiskPert (1%, 3%, 8%)
9
Infected embryos reach blastocyst
RiskUniform (12%, 14%)
stage
10
Infected embryos undetected by
RiskPert (2%, 3%, 20%)
laboratory tests
Risk associated with transferable embryo =
[(P1*P2*P3*P4) + (P5*P6*P7)]*P8*P9*P10
C-O-C: cumulus–oocyte complex
FMDV: foot and mouth disease virus
values from the probability functions at each node. The
analysis range shows all the possible outcomes, such as the
ones suitable for the most conservative, or intermediatelevel, decisions. This quantitative risk assessment (QRA)
model enables the regulatory authorities to compare the
acceptable level of risk with the likelihood of occurrence of
a scenario.
Estimation of the foot and mouth disease virus
risk at each step (or node) of the cloning pathway
Node 1. Is the selected donor
of the somatic cell likely to be infected?
Foot and mouth disease (FMD) is an acute vesicular
disease of cloven-hoofed domestic and wildlife species.
The disease, which is enzootic in several areas of the world,
including Africa, Asia, parts of South America, and the
Middle and Far East, is characterised by fever, loss of
appetite, salivation and vesicular eruptions on the feet,
mouth, and teats (39). Morbidity can reach 100% in
susceptible animal populations (2), although in vaccinated
populations only about 5%–10% of affected cattle herds
manifest the disease (35). This is the case in regions such
as South America, where vaccination often covers about
90% of each herd (36).
Pedigree animals, such as those that might produce the
donor cells that the cloning procedure is designed to
propagate, are normally subject to rigorous health
monitoring and surveillance during their lifetime (8, 45).
However, the clinical diagnosis of FMD can sometimes be
difficult, depending on the virulence of the virus and
vaccination status. The fact that the acute stage of infection
may be followed by prolonged, symptomless, persistent
infection (the carrier state) further complicates the
situation (2, 36). The proportion of such carriers depends
on the incidence of the disease (or infection) and the
immune status of the population, but carriers are found
relatively frequently in endemic areas. According to
Alexandersen et al. (1), 15%–20% of cattle and sheep in
Asiatic Turkey were found to be carriers. The same authors
quoted a carrier state of 50% in Brazil after a vaccine
breakdown. The proportion of animals that become
carriers under experimental conditions is variable but
averages around 50% (2, 36).
Based on the above evidence, the probability that a donor
of somatic cells is infected is estimated to equal the
proportion of persistently infected animals in enzootic
areas, i.e. 0.15 to 0.5 (most likely: 0.20) _______________P1.
Node 2. Would diagnostic tests fail
to detect an FMDV-infected donor of somatic cells?
Carrier cattle may continue to excrete virus for up to
3.5 years post infection. Carriers have also been shown to
mount cell-mediated and humoral immune responses and
to secrete virus-specific IgA in saliva (reviewed by Kumar
[17]). The gold standard test for FMD antibody detection
is the virus neutralisation test (VNT). However, when large
numbers of sera need to be tested, screening is usually
done by enzyme-linked immunosorbent assay (ELISA).
A solid-phase blocking ELISA for the detection of
antibodies directed against type O FMDV showed a
sensitivity of 99% (relative to the VNT) when used to test
148 positive cattle, goat and sheep sera collected from
FMDV-infected Dutch farms. The ELISA also correctly
scored 398 of 409 (97.3%) experimentally derived positive
sera (6). The Danish C-ELISA, which had a reported 92%
(35/38) sensitivity based on experimental infection, was
however only 71% sensitive at the recommended cut-off
when used to test samples from naturally infected cattle
(5).
Over the years, several ELISA techniques, mainly targeting
non-structural viral proteins (NSP), have been developed.
The initial estimates of the analytical sensitivity needed to
detect baculovirus-expressed FMDV non-structural
proteins 3AB and 3ABC were 73% (22/30) and 90%
(27/30), respectively. The 3AB and 3ABC antigens used in
this particular ELISA were also able to differentiate
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FMDV-infected pigs from vaccinated pigs (7). Kumar (17)
determined the sensitivity of a 3A-NSP-ELISA by testing
382 known positive sera; only three of these sera gave a
negative reaction (99.2% sensitivity). Nowadays, more
sensitive assays such as real-time polymerase chain
reaction (PCR) (97% sensitivity) have been developed to
detect the presence of viral RNA for all seven serotypes of
reference FMD viruses (17).
Based on the above evidence, the probability that screening
and confirmatory tests would fail to detect FMDV in the
infected donor of somatic cells is estimated to range from
0.01 to 0.29 (most likely: 0.03) _______________________P2.
Node 3. Would diagnostic tests fail to detect foot and
mouth disease virus in tissue culture?
Different cell culture systems, including pig cell line (IB-RS2) cells, baby hamster kidney (BHK-21) cells, Chinese
hamster ovary (CHO) cells, human mammary gland
epithelial cells, lamb kidney cells, Madin–Darby bovine
kidney (MDBK) cells and bovine thyroid (BTY) cells,
support the growth of FMDV (14, 17, 18, 28, 29). Given
that the virus is cytocidal, infected cells exhibit
morphological alterations, commonly called cytopathic
effects, which include cell rounding and alteration and
redistribution of internal cellular membranes (12). The BTY
cells show the best sensitivity for FMD virus detection (3).
According to Khawaja et al. (16), 42 of 62 known viruspositive epithelial suspensions (ES) had a cytopathogenic
effect on BTY cells. The same study quoted a report of 80%
viral detection from a previous study. Cultured cells can
also be assayed by ELISA (1, 16), fluorescent antibody
reaction (25), animal inoculation (24) or PCR (17, 23). In
general, the sensitivity of these tests ranges from 70%–80%
in cell culture, to 90% for ELISA and 97% for PCR.
Based on the above evidence, the probability that cell
cultures, animal inoculation, serology and PCR would fail to
detect FMDV in persistently infected somatic cells is
estimated to range from 0.02 to 0.2 (most
likely:0.03)____________________________________________P3.
Node 4. Would tissue culture treatment remove
foot and mouth disease virus from donor cells?
Several studies have shown that attachment of FMDV to
cells in cell culture requires specific receptors on the cell
surface. Accordingly, modification of these receptors can
inhibit attachment of the virus to cells. In this regard,
Jackson et al. (14) found that including heparin in the cell
culture completely inhibited virus attachment to fixed
cells; even attached viruses could be eluted by heparin.
Wild & Brown (43) had previously indicated that trypsininactivated FMDV failed to adsorb to pig kidney tissue
culture cells held in suspension, which suggests that
trypsin is able to remove or block a specific attachment site
on the protein coat of the virus.
Rev. sci. tech. Off. int. Epiz., 31 (3)
Similarly, O’Donnell et al. (28) exposed human mammary
gland epithelial cells to FMDV in the presence of
chlorpromazine, which inhibits clathrin-mediated
endocytosis, and assessed the number of virus-positive
cells after 4 h. They determined that there were about
50% FMDV-infected cells per field in the control cultures
compared with about 4% in the chlorpromazine-treated
cultures. La Torre et al. (18) treated monolayers of
BHK-21 cells lytically infected with FMDV, or line
C1-BHK-Rcl persistently infected with FMDV, with
increasing concentrations of ribavirin in the culture
medium. The ribavirin concentration that reduced the
virus yield to 50%, 24–48 h after addition of the drug, was
30 µg/ml to 50 µg/ml for the lytic infection and 3 µg/ml to
6 µg/ml for the persistent infection. Concentrations of
ribavirin of 150 µg/ml or higher resulted in a decrease in
virus production, and reduced infectious intracellular
RNA below detectable levels. Given that no renewed
FMDV production occurred during subsequent serial
passages, the authors concluded that treatment with
ribavirin had removed the FMDV from the persistently
infected Cl-BHK-Rcl cells. They also emphasised that
several of these ribavirin-cured cell lines have been derived,
passaged, frozen, thawed, and regrown by the same
procedures used for BHK-21 cells, without detectable loss
of viability.
Based on the above evidence, the probability that a somatic
cell culture line would still be infected after its treatment
during tissue culture with FMDV-inhibiting drugs is
estimated to range from 0 to 0.5 (most likely: 0.04)_____P4.
Node 5. Is the slaughtered oocyte donor infected?
As mentioned above, according to a review by
Alexandersen et al. (1), 15%–20% of cattle and sheep in
Asiatic Turkey were found to be carriers. The same review
quoted a carrier state of 50% in Brazil after a vaccine
breakdown. The proportion of exposed animals that
become carriers under experimental conditions is variable
but averages around 50% (2, 36).
Based on the above evidence, the probability that donors of
the oocytes are infected is estimated to equal the
proportion of persistent infection in enzootic areas,
i.e. 0.15 to 0.5 (most likely: 0.20)______________________P5.
Node 6. Could the cumulus–oocyte
complexes and the oocyte be infected?
Infection with FMDV is initially established in the noncornified epithelium of the pharyngeal area and then
spreads to regional lymph nodes and via the bloodstream
to cornified epithelial cells of the skin and mouth. All
secretions and excretions, including saliva, nasal and
lachrymal fluids, milk and expired breath, become
infectious. Urine and faeces also contain the virus (1).
Under natural conditions, 8 out of 30 viraemic cows
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(26.7%) had FMDV in their reproductive tracts (24). The
virus is highly concentrated in the follicular fluid and
ovarian tissues, although the ovaries of convalescent cows
killed six weeks after being diagnosed with the disease
were not infectious (22), signifying that FMDV does not
persist for long in the ovaries of infected cows.
Based on the above evidence, the probability that C-O-Cs
could be contaminated in the reproductive tract of females
(persistently) infected with FMDV is estimated to range
from 0 to 0.27_________________________________________P6.
Node 7. Would the processes of in vitro maturation
and washing remove foot and mouth disease virus
from the oocyte/cumulus–oocyte complexes?
Mebus & Singh (24) used in vivo uterine flushing to collect
a total of 436 embryos and unfertilised ova from
FMD viraemic cows. After appropriate washing (according
to the International Embryo Transfer Society [IETS]
protocols), degenerated embryos (n = 144) and unfertilised
ova (n = 64) were assayed either in cell culture or by
inoculating them intradermally into the tongues of steers.
None of the sonicated ova and embryos was found to be
associated with the virus. Similarly, Jooste (15) collected
C-O-Cs from the ovaries of slaughtered cows and exposed
them to FMDV by adding the virus to the maturation
medium. Although the FMDV-exposed C-O-Cs and
cumulus cells produced cytopathic effects on pig kidney
cells even after washing, oocytes that had been denuded of
cumulus by treatment with morpholino-ethanosulphate
(MES) were negative for the virus when tested by cell
culture and by PCR.
Based on the above evidence, the probability that in vitrocultured, denuded, washed, and treated oocytes would carry
FMDV is estimated to range from 0 to 0.05______________P7.
Node 8. Would embryo cleansing
by washing remove foot and mouth disease
virus from the developing embryo?
Although 8 out of 30 FMD-viraemic cows (26.7%) had the
virus in their reproductive tracts (24), the intact ZP,
especially that of in vivo-derived embryos, acts as a barrier
for the majority of infectious agents, including FMDV (36).
It has also been shown that FMDV does not adhere firmly
to the intact ZP of in vivo-derived embryos (31, 38),
provided that the latter are properly washed according to
IETS protocols (34). To investigate whether FMDV
interacts differently with bovine embryos produced in vitro,
a suspension of FMDV was added to batches of seven-dayold in vitro-fertilised embryos which were then incubated
with virus for periods of 1 h, 2 h or 4 h. After the embryos
had been washed ten times, they were pooled and ground
up to form a suspension, and this suspension was then
assayed on cell cultures for FMDV. Assays for FMDV were
also conducted on the first and second wash and on the
pooled sample constituting the eighth, ninth and tenth
washes by cell culture and PCR. Although almost all
samples of the first and second wash, and both developed
and degenerated embryos (8 out of 12 batches), produced
FMDV cytopathic effects, all pooled eighth to tenth washes
were cytopathogenically negative (23).
Similarly, Singh et al. (31) exposed 169 in vivo-derived
ZP-intact bovine embryos to 106 plaque-forming units
per/ml of FMDV. After standard washing, no infectious
virus was detected on any of the embryos. However, FMDV
infectivity was found in association with 14 of 42 hatched
(ZP-free) bovine embryos and four of the 124 ZP-intact
porcine embryos. It was noted that the level of infectivity
did not increase with further incubation, which suggests
that replication of the virus in embryonic cells is unlikely.
In a later study (24), a total of 653 in vivo-derived bovine
embryos/ova were exposed to FMDV in vitro or by
collecting them from FMDV-inoculated animals. These
embryos/ova were washed and then tested for FMDV
infectivity. The virus was not found to be associated with
any of the embryos/ova. Any residual infection could be
removed by adding trypsin to the embryo wash fluids; this
inactivates FMDV by removing or blocking a specific
attachment site on the protein coat of the virus (43).
Based on the above evidence, the probability that embryo
clones could still carry FMDV after proper washing and
trypsin treatment is estimated to range from 0.01 to
0.08 (most likely: 0.02) _______________________________P8.
Node 9. Would embryos infected with foot and mouth
disease virus develop into exportable blastocysts?
In a study by Jooste (15), it was found that 18.8% (47/250)
of in vitro-produced bovine embryos exposed to FMDV
in vitro reached blastocyst stage, compared with 22.2%
(55/250) for the non-exposed control embryos. Using
typical culture conditions, it was found that 105 of
569 fused couplets (18.5%) developed to bovine cloned
blastocyst stage (46).
Based on the above evidence, the probability that FMDVinfected cloned embryos would develop into transferable
blastocysts is estimated to range from 0.12 to 0.14 ____P9.
Node 10. Would foot and mouth disease
virus infection fail to be detected
in the developed transferable blastocyst?
Laboratory diagnosis of FMDV in embryos and wash fluids
is usually made by detection of specific FMDV antigens
using ELISA (15), cell culture (22, 23, 24, 31), animal
inoculation (22, 24, 31), or PCR (15, 23), and the
sensitivity of these techniques is probably reasonably
comparable with their sensitivity in tissue samples. In one
study (23), 8 of 12 batches of first and second wash fluid
samples from experimentally contaminated embryo
cultures produced cytopathic effects. In general, the
sensitivity of these tests ranges from 70%–80% in cell
768
culture (16), 70%–90% in ELISA (1) and 70%–97% in
PCR (17).
Based on the above evidence, the probability that
diagnostic tests would fail to detect FMDV-infected
embryos or FMDV-contaminated wash fluids is estimated
to range from 0.03 to 0.10 ___________________________P10.
Effect of the number
of imported embryos on the estimated risk
Most in vitro systems use oocytes collected either from the
ovaries of slaughtered cows or by ovum pick up (OPU).
On average, it is possible to recover up to 12 good quality
oocytes from a slaughtered cow (15). Using OPU, on the
other hand, Yang et al. (46) obtained 8.2 C-O-Cs per
heifer. The maturation and fusion rates of bovine oocytes
were 81% (1,100/1,357) and 63% (569/906), respectively.
A good proportion, i.e. 450 of 569 fused couplets (79%),
were successfully activated and cultured, leading to a 67%
(300/450) cleavage rate and 23% (105/450) development
to blastocyst stage. According to the same study, 4 of
18 transferred blastocysts (22.2%) resulted in pregnancy;
the calving rate was 6% (1/18) (46). Forsberg et al. (9)
previously reported 59% (10/17) and 30% (3/10)
pregnancy initiation and calving rates, respectively, using
adult fibroblast-like cells as the donor cells for the
production of cloned embryos. On the other hand, Wells
(41) reported a pregnancy rate of about 65%, although
only 13% (n = 988) of transferred cloned embryos resulted
in calves delivered at full term.
Assumptions made
Although the systems used for the production of cloned
embryos are very diverse, procedures for managing animal
health risks associated with cloned embryos have been
described (45). However, specific information regarding
FMDV transmission through cloned embryo production
and transfer is lacking. For this study, therefore, relevant
information regarding the possible interaction of FMDV
with cloned embryos had to be extrapolated, mainly from
studies conducted on the interactions of FMDV and other
pathogens with bovine oocytes and embryos. In particular,
the research focused on the potential for embryo
contamination either from somatic cell or oocyte donors.
As a valuable pedigree animal, a donor of somatic cells will
almost certainly have been subjected to health monitoring
and surveillance (8, 45). Oocytes from which the recipient
cytoplast is produced are, on the other hand, collected
from the ovaries of randomly selected females slaughtered
at commercial slaughterhouses (15). Given that there are
no specific new risks identified with SCNT cloning (45),
the primary animal health risks are associated with the
health status of the animal from which the ovaries are
Rev. sci. tech. Off. int. Epiz., 31 (3)
harvested. For this reason, the potential risks (and the
respective risk mitigations) for the somatic cells and
oocytes have been dealt with separately, prior to their
fusion to produce the cloned couplet. The potential risk
after the fusion has been assessed by reference to
internationally accepted protocols for processing embryos
(34) and to the results of testing degenerated embryos and
wash fluids for FMDV. The risk of introduction of
extraneous infectious agents such as FMDV, from
supporting cells and animal products such as serum, was
not addressed in this study. It is assumed that the cloning
industry will have observed the internationally agreed
recommendations to ensure that media, reagents and
equipment, as well as the working environment, are free of
pathogens and contaminating microorganisms (33).
Whether this assumption is justified is difficult to
ascertain, but it should be judged by the regulatory
authorities in the USA.
The number of imported cloned embryos was initially
suggested to be 100 per year. This assumption takes into
account the number of companies engaged in cloning and
various applications of cattle cloning, which include
improving disease resistance (41). Given that the success of
cloning is measured by having a live offspring (and a
surviving dam), the efficiency of the cloning procedure is
critical in determining the number of imported cloned
embryos. Based on these facts, and in order to provide a
realistic risk estimate, a yield of between 10 and
50 (mean = 30) cloned offspring per year was assumed.
The number of imported cloned embryos, which ranges
from 250 to 1,700 (mean = 520), was thus obtained by
modelling contingent on the range of clones produced per
year, rather than using a fixed number of imported cloned
embryos as originally suggested (details are provided in
Table II).
Results
In this study, critical pathway analysis was applied to
evaluate the risk of introducing FMDV into the USA via
cloned bovine embryos created in endemically infected
regions outside the USA. Specific inputs for the model and
respective calculations are displayed in Tables I and II. A
summary of the results is displayed in Table III. The risk
estimate (3.1 × 10–7) and the probability density/
cumulative probability distribution are shown in Fig. 3 for
a better graphical presentation. Assuming the importation
of 250 to 1,700 cloned embryos per year, contingent on
producing 10 to 50 cloned offspring, and using the data
given in Tables I and II as inputs, the expected number of
FMDV-infected cloned embryos imported into the USA per
year is 1.6 × 10–4 (Fig. 4). Correspondingly, Fig. 5 depicts
the number of years (5.5 × 104) it would take before an
769
Rev. sci. tech. Off. int. Epiz., 31 (3)
Discussion
Table II
Summary of parameters for estimating the number of
transferable embryos
Probability
Description
Distribution
A
No. of oocytes per cow/heifer
B
Maturation rate of bovine oocyte RiskBeta
RiskUniform (8, 12)
(1100+1,(1357–1100)+1)
C
Fusion rate of mature oocyte
D
Cleavage rate of fused oocyte
RiskBeta (569+1,(906–569)+1)
RiskBeta (300+1,(569–300)+1)
E
Blastocyst development rate
RiskBeta (105+1,(300–105)+1)
of cleaved oocyte
F
Pregnancy rate of embryo
G
Pregnancy yielding live calf (clone) RiskUniform (25.0%, 30.0%)
RiskPert (13.0%, 22.2%, 65.0%)
H
Live clones produced per year
RiskPert (10, 30, 50)
Number of embryos imported per year = [1/(F*G)]*H
FMDV-infected cloned embryo is imported into the USA
(details are provided in Table III). According to the
sensitivity analysis (Fig. 6), the inputs showing the highest
variability/uncertainty were: the FMDV status of the C-OCs, the effectiveness of the embryo washing protocol and
embryo testing procedures, and the FMDV status of oocyte
donors.
The infectious hazards posed by cross-border movement of
cloned embryos to the livestock industry, and in some
cases to human health in the importing country, have not
been well elucidated. This makes regulatory authorities
uncomfortable with the international trade of cloned
embryos between nations. Nevertheless, QRA is a logical
approach that could provide these authorities with
plausible information on which to base their decisions. In
this study, the authors applied QRA methodology to
evaluate the risk of introducing FMDV into the USA via
cloned embryos imported from regions where FMD is
endemic. As shown in the results section, the risk is very
small, less than 3.1 × 10–7. This is mainly due to the
beneficial effect of embryo washing procedures and the
efficiency of diagnostic tests. Oocyte/embryo washing,
especially if combined with the use of trypsin, can easily be
incorporated into SCNT techniques and has been shown to
limit FMDV transmission effectively when conventionally
produced embryos, either exposed to the infectious agent
in vitro or collected from infected donors, were transferred
into disease-free recipients (33). In fact, digestion of the
source tissues with trypsin is a routine procedure in tissue
culture (25) and cattle cloning (9, 10, 42). Trypsin is
known to inactivate viral particles attached to the ZP
(33, 45). The assessment therefore substantiates earlier
Table III
The results of 50,000 iterations showing: 1) probabilities at each node, and 2) the probability of introducing foot and mouth disease
virus into the USA, the number of cloned embryos imported per year, the number of infected embryos imported per year, and the
number of years it would take for an infected cloned bovine embryo to be imported into the USA
Probabilities
Minimum
5th percentile
Mean
95th percentile
Maximum
P1
1.5 10
1.6 10
2.4 10
3.5 10
4.8 10–1
1.7 10
7.0 10
–1
P2
1.1 10
1.6 10
2.8 10–1
P3
P4
2.4 10–5
1.0 10–2
1.1 10–1
2.7 10–1
4.7 10–1
2.0 10–2
2.0 10–2
6.0 10–2
1.1 10–1
1.8 10–1
P5
1.5 10
1.7 10
3.3 10
4.8 10
5.0 10–1
P6
1.9 10
1.0 10
1.4 10
–1
2.6 10
2.7 10–1
P7
2.9 10–7
2.5 10–3
2.5 10–2
4.8 10–2
5.0 10–2
P8
1.0 10–2
1.6 10–2
3.5 10–2
5.8 10–2
7.8 10–2
P9
1.2 10–1
1.2 10–1
1.3 10–1
1.4 10–1
1.4 10–1
P10
–2
2.0 10
2.4 10
5.7 10
1.1 10
1.8 10–1
Probability that an
imported embryo
is contaminated
–10
3.8 10
1.7 10
3.1 10
–6
1.0 10
4.6 10–6
No. of cloned embryos*
imported per year
2.5 102
2.7 102
5.2 102
9.6 102
1.7 103
No. of infected embryos
per year
1.1 10–7
7.3 10–6
1.6 10–4
5.6 10–4
4.4 10–3
Years until infected
embryo is imported
5.3 102
3.1 103
5.5 104
2.0 105
6.9 106
–1
–2
–1
–6
–1
–2
–1
–2
–2
–8
*Contingent on the assumption of producing 10 to 50 cloned offspring per year
–1
–2
–1
–1
–2
–7
–1
–1
–1
770
Rev. sci. tech. Off. int. Epiz., 31 (3)
5%
100
90
80
70
60
50
40
30
20
10
0
2.5
0.02
0
0.5
1
1.5
2.0
Cumulative frequency %
0.0045
0.004
0.0035
0.003
0.0025
0.002
0.0015
0.001
0.0005
0.
90%
Mean = 3,107E-007
Relative frequency
5%
Probability (in millionths)
0.005
0.0045
0.004
0.0035
0.003
0.0025
0.002
0.0015
0.001
0.0005
0
90%
5%
0.008
5%
0.565
Mean = 0.000160
Relative frequency
Fig. 3
Probability density function (cumulative distribution) for the risk of introducing foot and mouth disease virus into the USA via cloned
embryos created outside the USA
0
0.2
0.4
0.6
Number of infected embryos (in thousandths)
0.8
1
1.2
Fig. 4
Probability density function for the number of embryos infected with foot and mouth disease virus imported into the USA per year
5.0%
0.0%
+
95.0%
3.41
0.08
0.06
Mean = 4.2424
Relative frequency
0.1
0.04
0.02
0
2.25
2.75
3.25
3.75
4.25
Number of years (Log. transformed)
4.75
5.25
5.75
6.25
Fig. 5
Possible number of years before foot and mouth disease virus is introduced into the USA via importation of cloned embryos
C-O-C
Oocyte washing
Embryo testing protocols
Embryo washing
Oocyte donor
Cell culture treatment
Diagnostic test efficiency
Cell testing protocols
Embryo development
Cell donor
0.43
0.43
0.39
0.29
0.23
0.05
0.05
0.04
0.04
0.02
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Coefficient value
C-O-C: cumulus–oocyte complex
Fig. 6
Regression sensitivity analysis for the likelihood of introduction of foot and mouth disease virus into the USA via cloned embryos
771
Rev. sci. tech. Off. int. Epiz., 31 (3)
work demonstrating that conventionally produced (in
vitro-developed) bovine embryos, which may still contain
FMDV after a regular washing protocol (23), can be
rendered free of virus by exposure to trypsin (33, 37) or
other acids (15) in the culture media.
It has been shown above that the risk of transmitting
FMDV is significantly affected by the status of C-O-Cs and
their handling. This study may have erred towards an overcautious approach to levels of risk in the somatic cell and
C-O-C/oocyte donors. However, most abattoirs from
which C-O-Cs/oocytes are obtained perform thorough
ante-mortem and post-mortem inspections, so viraemic
(hence febrile) and clinically ill animals are unlikely to be
selected as oocyte donors. In addition, as far as the authors
are aware, FMDV has never been found in the ovaries or
C-O-Cs of chronically infected animals (17, 22).
Furthermore, sensitive tests can be used to detect FMDV in
the donors and in any degenerated embryos and wash
fluids, so that if infection is present the cells and oocytes
are not used for cloned embryo production. It should also
be noted that in persistently infected donor animals
(‘carriers’), the FMDV is usually located in the dorsal soft
palate and in the pharyngeal roof above the soft palate
(17). For SCNT procedures in cattle, differentiated muscle
cells (10) or fibroblasts obtained from skin or the ear
punch (9, 41, 42) tend to be used, and so high-risk
palatine tissues can be avoided.
Taken together with the published results of experiments
involving in vivo and in vitro exposure of bovine embryos
to FMDV, this work suggests that the risk of transmitting
the virus via the importation of cloned embryos is
negligible, even if the donors of the somatic cells and/or
the oocytes are persistently infected at the time of
collection. The IETS has classified FMD as a Category-1
disease, meaning that the risk of transmission via in vivoderived bovine embryos is negligible, provided that
embryos are handled according to IETS protocols between
collection and transfer (44). Thus, proper processing of
cells, oocytes and embryos (including exposure to trypsin),
coupled with practices that ensure that donors are not
acutely infected when cells are collected, should render
cloned embryos safe even if imported from FMD-affected
regions.
As far as the authors are aware, QRA has not been applied
previously to analyse the risk of introducing FMDV via the
movement of infected cloned embryos. However, the
World Organisation for Animal Health (OIE) has also
concluded that international movement of cloned embryos
is unlikely to lead to the release of adventitious infectious
agents (45). The OIE did note that the extensive
manipulation of donor cells, oocytes and embryos that is
involved in cloning by SCNT may exacerbate the risk of
transfer of adventitious agents and that the risks from
cloning are greater than those involved in in vitro
fertilisation (IVF) procedures. In order to further minimise
any perceived risk associated with cloning by SCNT,
practitioners should espouse the IETS recommendation
that the media in which the embryos are manipulated
before compromising the integrity of the ZP should be
sterile (37). If this can be attained, embryo production by
in vitro techniques has low potential for transmitting
diseases (33, 36), and this can be applied in evaluating
risks in SCNT (45).
Embryo production by SCNT (and IVF) also has one major
comparative advantage. The production pathway provides
control points and sufficient time to allow for each batch of
embryos produced to be monitored and assessed for their
health status. Such control begins with the strict
maintenance of medical records for the donor of the somatic
cells. Furthermore, regular sampling of all the media used in
the process and any degenerated embryos would give a very
accurate indication of the infectious milieu to which viable
embryos may have been exposed (36).
In conclusion, according to this QRA, the risk of
introduction of FMDV into the USA via cloned bovine
embryos is negligible and in the order of 3.1 × 10–7.
However, to provide further experimental evidence to
substantiate this conclusion, it would be necessary to use
somatic cells and oocytes from infected donors to produce
cloned animals and transfer them into recipients.
Acknowledgements
This study was supported by funds provided by the United
States Department of Agriculture, Animal and Plant Health
Inspection Service through a cooperative agreement to
Tuskegee University, Tuskegee, Alabama.
772
Rev. sci. tech. Off. int. Epiz., 31 (3)
Évaluation quantitative du risque d’introduction du virus de la
fièvre aphteuse aux États-Unis d’Amérique lors d’importations
d’embryons bovins clonés
B. Asseged, B. Tameru, D. Nganwa, R. Fite & T. Habtemariam
Résumé
La sécurité des échanges internationaux d’animaux d’élevage ou de leurs
produits s’appuie sur les informations obtenues concernant le risque
d’introduction des agents infectieux, en particulier le virus de la fièvre aphteuse.
Bien que les voies de transmission de ce virus puissent varier, l’existence d’un
risque d’introduction lié aux importations d’embryons clonés a récemment été
suspectée aux États-Unis d’Amérique. En conséquence, un modèle d’évaluation
quantitative du risque a été élaboré afin de déterminer, au moyen d’un calcul des
probabilités, les scénarios pouvant donner lieu à l’introduction puis au maintien
du virus de la fièvre aphteuse suite à l’importation d’embryons bovins clonés. La
probabilité d’introduction du virus de la fièvre aphteuse par des embryons
clonés, telle qu’elle a été estimée au moyen du logiciel @RISK associé à une
simulation de Monte-Carlo portant sur 50 000 itérations, s’est élevée à 3,1 × 10–7.
Compte tenu des protocoles en vigueur pour le clonage, et dans l’hypothèse
d’une importation annuelle de 250 à 1 700 embryons clonés (moyenne = 520), le
nombre escompté d’embryons infectés varie de 1,1 × 10–7 à 4,4 × 10–3 (moyenne
= 1,6 × 10–4) par an. L’analyse des voies critiques de transmission a montré que le
risque d’introduction du virus de la fièvre aphteuse aux États-Unis par cette voie
est extrêmement faible.
Mots-clés
Analyse des voies critiques de transmission – Clonage – États-Unis d’Amérique –
Évaluation quantitative du risque – Fièvre aphteuse – Simulation de Monte-Carlo.
Evaluación cuantitativa del riesgo de introducción
del virus de la fiebre aftosa en los Estados Unidos
de América a través de embriones bovinos clonados
B. Asseged, B. Tameru, D. Nganwa, R. Fite & T. Habtemariam
Resumen
El comercio internacional de ganado bovino y sus derivados exige disponer de
datos sobre el riesgo de introducción de agentes infecciosos como el virus de la
fiebre aftosa (VFA). Aunque este virus puede transmitirse por diversas vías, en
los Estados Unidos de América (EE.UU.) preocupa últimamente la posibilidad de
que penetre en el país a través de embriones clonados. Los autores describen
un modelo de evaluación cuantitativa del riesgo elaborado con el fin de
determinar (con su correspondiente probabilidad matemática) las eventuales
situaciones que podrían desembocar en la introducción y permanencia del virus
a consecuencia de la importación de embriones bovinos clonados. Utilizando el
programa informático @RISK con una simulación de Montecarlo que entraña
50.000 iteraciones, se cifró en 3,1 × 10–7 la probabilidad de introducción del VFA
773
Rev. sci. tech. Off. int. Epiz., 31 (3)
a través de embriones clonados. Habida cuenta del protocolo actualmente
aplicado, y presuponiendo un volumen de importaciones anuales de entre 250 y
1.700 (mediana = 520) embriones clonados, se calcula que el número anual de
embriones infectados se sitúa entre 1,1 × 10–7 y 4,4 × 10–3 (mediana = 1,6 × 10–4).
El análisis de rutas críticas dejó patente un riesgo extremadamente bajo de
penetración del VFA en los EE.UU. por esta vía.
Palabras clave
Análisis de rutas críticas – Clonación – Estados Unidos de América – Evaluación
cuantitativa del riesgo – Fiebre aftosa – Simulación de Montecarlo.
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