D9133.PDF

Rev. sci. tech. Off. int. Epiz., 16 (1), 226-239
The risks of disease transmission by embryo
transfer in cattle
(1)
P. Sutmoller & A.E. W r a t h a l l
(z)
(1) Pan American Foot and Mouth Disease Center, Caixa Postal 589, CEP 20001-970 Rio de Janeiro, Brazil
[2) Central Veterinary Laboratory, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom
Summary
Guidelines for the safe international movement of livestock embryos are provided
in the International Animal Health Code of the Office International des Epizooties,
and recommendations for embryo processing, based on numerous research
papers on embryo-pathogen interaction studies, are given in the Manual of the
International Embryo Transfer Society. Risk assessment is the logical extension of
these approaches, since it provides veterinary authorities with a complete
package of information on which to base their import/export decisions. Risk
assessment includes evaluation of disease prevalence, effectiveness of
Veterinary Services and competence of the embryo collection team. It also takes
account of the epidemiology and pathogenesis of the disease concerned.
The application of risk assessment for embryo movement is illustrated in this
paper by comparisons of the probabilities of transmitting foot and mouth disease,
bluetongue and vesicular stomatitis by bovine embryos. The risk scenario
pathway was divided into three phases for analysis. The first phase deals with the
potential for embryo contamination, which depends on the disease situation in the
exporting region, the health status of donor herds and donor cows, and on the
pathogenetic properties of the disease agent. The second phase covers risk
mitigation by use of the internationally accepted standards for embryo
processing, and the third phase considers the risk reductions resulting from
post-collection surveillance of donors and donor herds, and also from testing of
embryo-collection (flushing) fluids for the disease agent.
It was evident from this assessment that low risks of transmitting disease by
international movement of bovine embryos depend initially on a low disease
incidence in the exporting region and on easily recognisable disease signs.
Competent embryo processing was also of great importance, and in the case of
bluetongue, vector ecology had a major influence.
In addition to providing a logical basis for import/export decisions, risk
assessment is useful for evaluating the potential outcome of new research and for
assessing the safety of the movement of embryos of other species for which little
or no research information is available on embryo-pathogen interactions.
Keywords
Cattle - Disease transmission - Embryos - Importation - Risk assessment.
Introduction
The range of infections which conceivably might be
transmitted by embryo transfer is vast, but it appears that the
risks are far lower than those associated with movement of
postnatal animals, semen and most other animal products.
This conclusion is based not only on a large amount of
research, but also on field experience. For example, more than
1,000 bovine embryos were imported into France from the
United States of America (USA) between 1 9 8 3 and 1987
without any prior testing of the donors or even washing of the
embryos, yet no evidence of disease transmission was detected
(37). The recent importation of over 8,000 goat embryos into
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Rev. sci tech. Off. int. Epiz., 16 (1)
Canada from South Africa provides another example: whilst
some pre-export testing of the donors was performed in this
instance, there has been no evidence of embryo-associated
disease transmission ( 9 ) . Surveys conducted for the
International Embryo Transfer Society (IETS) over the past
few years ( 3 6 ) indicate that numbers of bovine embryos
transferred amount to more than 3 5 0 , 0 0 0 per annum, of
which about 1 0 % are moved internationally. Numbers of
transferred sheep, goat and swine embryos are much lower
than those of cattle, but are not insignificant. A high
proportion of the transfers, particularly within countries, are
made without accurate knowledge of the health status of the
donors, so it is noteworthy that there have been no
substantiated reports anywhere in the world of disease
transmission to recipients by transfer of an embryo. Despite
this outstanding safety record, there are still good reasons to
be cautious, because there are no non-destructive methods
available to test embryos for freedom from pathogens. There is
also the possibility of transmitting pathogens via
contaminated media, or items of equipment used to collect,
wash, freeze and thaw embryos. Biological constituents of
media (e.g. serum or serum albumen), and also hormones and
enzymes such as trypsin, give rise to special concerns about
their inherent disease status ( 1 , 4 , 13). Searches for safer
alternatives are progressing ( 2 0 , 2 5 ) , but meanwhile any
materials of animal origin which are used must originate from
safe sources and they must be properly processed to remove
potential pathogens and contaminants.
Bovine embryos for international trade
Most bovine embryos which are traded internationally are
obtained from donor cows which have been superovulated
using hormone treatments and then artificially inseminated
(AI).
Such in vivo derived embryos are recovered
non-surgically seven days after AI by flushing the uterus with
A: Zona pellucida-intact morula
B: Zona pellucida-intact blastocyst
C: Zona pellucida-intact expanding blastocyst
Fig. 1
Bovine embryos at different stages of development
Embryos D, E and F are non-exportable in terms of disease transmission
Source: Manual of the International Embryo Transfer Society (IETS) (32)
collection medium. The embryos and uterine debris are
separated from the rest of the medium either b y
sedimentation or filtration. The sediment or filtrate is
examined systematically at low magnification in order to
locate the embryos. The embryos are then transferred to small
dishes with fresh medium and evaluated according to the
IETS Manual (32).
The
zona pellucida (ZP) is an important barrier against
pathogens,
so
only
ZP-intact
viable bovine
embryos,
including ZP-intact expanding blastocysts should be exported
(Fig. 1).
Bovine embryos considered non-exportable in terms of their
disease transmission risk are those with a broken or otherwise
defective ZP or with extraneous material adhering to the ZP,
as well as blastocysts which have hatched and which therefore
do not have a ZP ( 3 2 ) . Embryos destined for international
movement must be 'washed' at least ten times. For this
procedure, they are transferred through a series of ten dishes
or wells, each containing approximately 2 ml of medium.
Each 'wash' must constitute at least a one hundred-fold
dilution of the medium in the pipette with which the embryos
are moved from one dish to the next, and a new, sterile pipette
must be used for each of the washes. The embryos are kept
under low power microscopic observation and gentiy agitated
during the washings. They are finally observed at higher
magnification to ensure that the ZP is still intact and free from
extraneous cellular debris. This procedure is described in
detail in the IETS Manual ( 3 2 ) . Embryos are normally kept
frozen from the time of collection until exportation and use.
Disease concerns
Time spent by in vivo derived embryos in the oviduct or
uterus before collection is only a few days. There is a
D: Morula with a defect in the zona pellucida
E: Morula with extraneous material adhering to the zona pellucida
F: Hatched blastocyst without a zona pellucida
228
possibility that embryos from infected donors may have
encountered infectious agents whilst in these sites, but this
depends to a large extent on the pathogenesis of the agent.
Predilection for the genital tract is of special relevance with
respect to transmission by embryo transfer. Among the many
known genital diseases of livestock are brucellosis, bovine
genital campylobacteriosis and trichomoniasis. It is customary
to include infectious bovine rhinotracheitis (IBR) and other
infections such as Haemophilus somnus, Leptospira,
Chlamydia
and genital Mycoplasma
in this list. Pantropic and
blood-borne agents (of which there are many) may readily
gain access to the genital tract and also to the embryos. With
other diseases, the risk of embryos having contact with the
infectious agent under normal circumstances is remote, and
here those which tend to solely infect skin might be included
(e.g. poxviruses and dermatomycoses), or gut (e.g. rotaviruses
and enterobacteria). However, such agents do occasionally
produce generalised infections, and even when very localised
there is always a possibility of the embryo collection media
and equipment becoming contaminated.
The nature of a disease, especially its epidemiology and
potential economic impact on livestock populations, is a
factor of paramount importance to veterinary authorities
when gauging threats to importing countries ( 1 0 ) . For
example, agents such as foot and mouth disease (FMD) and
rinderpest can pose enormous threats, and measures to avoid
them must be commensurate. On the ©ther hand, there might
seem little point in imposing strict measures to avoid
introduction of diseases which are already endemic in the
importing country, especially if no national control policy
exists. The exclusion of new strains of endemic disease agents
does warrant some attention, however, because these could be
more virulent than the indigenous agents, not covered by
vaccines, and their spread might cause major losses.
Certain diseases are particularly worrying to veterinary
authorities because of the special diagnostic problems
encountered
when
certifying
that
embryos
are
specific-pathogen free. The so-called 'slow virus' infections,
such as bovine spongiform encephalopathy (BSE) in cattle, are
listed in this category because there are no serological or other
tests to identify subclinically infected and incubating animals
(40). Finally there are common commensal bacteria and the
ubiquitous contaminants which may be picked up during
embryo collection and processing procedures: while perhaps
not posing a direct threat, their identification in samples of
flushing and washing media can be a useful indicator of poor
sanitary procedures (38).
Animal health protocols for international
movement of embryos
Early contacts (21) between the IETS and the Office
International des Epizooties (OIE) led to collaboration in
producing guidelines for the safe international movement of
Rev. sci tech. Off. int. Epiz., 16 (1)
bovine, ovine/caprine and porcine embryos, which were
subsequently published by the OIE as Appendices in the
International Animal Health Code (22). The OIE International
Animal Health Code, together with the OIE Manual of
Standards for Diagnostic Tests and Vaccines ( 2 3 ) , contains
recommended rules and test procedures for the international
movement of animals and animal products, and these are
recognised by the veterinary authorities of most governments.
A fundamental concept enshrined in the relevant Appendices
of the OIE International Animal Health Code ( 2 2 ) is the
accountability of the 'Officially Approved Embryo Collection
Team' which operates under the supervision of the 'Team
Veterinarian'. Approved embryo collection teams in exporting
countries are held responsible for adhering to all official
sanitary protocols for embryos, including collection,
processing, identification, grading and certification before
export, details of which are covered in the IETS Manual (32).
Major benefits of the joint OIE/IETS approach are that when
the recommended protocols are followed, not only can the
imported embryos be considered safe, and certified as such,
but the costly multiple testing regimes which were formerly
stipulated for donors and herds of origin in the exporting
country, and the quarantine systems sometimes used for
recipients and embryo transfer (ET) offspring in the importing
country can usually be dispensed with.
The Import/Export Committee of the IETS has categorised
several disease agents as 'agents for which sufficient research
evidence has accrued to show that the risk of disease
transmission by embryo transfer is negligible provided that
between collection and transfer the embryos are handled as
recommended in the IETS Manual'
( 5 ) . While this
information can be helpful to veterinary officials who make
decisions on import risks, the system does have weaknesses.
For example, FMD and IBR are both listed in category 1, but
for veterinary officials 'negligible' applied to a catastrophic
disease like FMD has a very different meaning from when it
refers to a commonly endemic disease such as IBR. Obviously,
the socio-economic consequences of inadvertent introduction
of FMD are far greater than those for IBR, and officials must
take account of not only the level of risk of disease
introduction, but also the potential impact of the disease on
national livestock populations. To expect regulatory officials
to allow unregulated international movement of embryos
merely because certain diseases are listed in category 1
('negligible risk of transmission') would obviously be naive. In
fact, until recently many countries have applied a 'zero risk
policy with respect to importation from regions where severe
epidemic diseases exist, and it is unlikely that this ultra-safe
policy will be abandoned without extreme caution. Another
possible reason for the apparent reluctance of some national
veterinary authorities to relax requirements for importation of
embryos may have been that the IETS Import/Export
Committee did not define precisely what was meant by the
term 'negligible risk'.
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Rev. sci tech. Off. int. Epiz., 16 (1)
Risk assessment of disease
transmission by embryos
'Risk', in relation to importation of embryos, is a measure of
the probability of introduction of an exotic disease and the
seriousness of such an outcome. 'Risk assessment' is the
process of estimating, as objectively as possible, the
probability that an importation would result in the entry of an
exotic disease agent, and that indigenous livestock in the
importing country would be exposed. 'Risk management' is
the process whereby risk is reduced to an acceptable level by
appropriate strategies ( 2 ) .
Consistent, reliable and scientifically based risk assessment is
an essential ingredient for effective risk management, and the
discipline of 'quantitative risk assessment' (QRA) is
increasingly being applied. QRA has been used for many years
in engineering and economics, but it is rapidly gaming
acceptance in the veterinary profession as a basis for
regulatory actions, including those connected with
importation of animals and animal products, (7, 1 1 , 15, 16,
18, 19, 2 4 ) . QRA involves the use of epidemiology,
microbiology, pathology, economics and statistics, in addition
to common sense, to give precise mathematical estimates of
risk, thereby enabling importation conditions that are
acceptable and justified. As the chances of transmitting
diseases by embryo transfer are so low and because research
results (especially those from in vivo experiments) have
usually been negative, QRAs are, of necessity, based on
statistical probabilities.
'Scenario pathway analysis' ( 1 2 ) is a methodology which is
especially appropriate for quantitative evaluation of the risks
associated with importation of animals or animal products.
For example, in the case of embryo imports, every
risk-associated event in the pathway is itemised from the
point of origin to final destination of the embryos. The
probability or frequency with which such events might occur
is evaluated. Accumulation of the numerical answers to these
evaluations enables a value to be assigned to the risk
associated with the entire pathway. The end result is a
quantitative measure of risk that can be clearly visualised and
for which the evidence is fully documented.
Recent risk studies applicable to embryo
transfer
Sutmoller ( 3 3 ) discussed some of the disease transmission
risk factors related to embryo transfer, and Sutmoller and
Wrathall (34) attempted to quantify levels of FMD risk
reduction which will be achieved by adhering to the IETS
Manual recommendations ( 3 2 ) . An important conclusion
emerging from those studies was that there are three main
lines or levels of defence against the introduction, of diseases
via embryo transfer. The first defence line encompasses an
evaluation of the disease risk situation in the exporting
country and/or region, the health status of the farms and
donor cows from which the embryos are collected, and the
pathogenic characteristics of the specified disease agent. The
second line depends on the use of accepted standards of
handling and processing of the embryos (32). The third line of
defence includes any post-collection surveillance of the
donors and donor farms, and also the possible testing of
embryo collection (flushing) fluids for presence of the disease
agent.
Scenario pathway analyses have recently also been used to
quantify and compare the risks of transmission of FMD,
bluetongue (BT) and vesicular stomatitis (VS) by bovine
embryos imported from a region where these three diseases
exist ( 3 5 ) . Account was taken of the very different
characteristics of those diseases. FMD, with its catastrophic
effects, was an obvious choice for study; its pathogenicity is
well understood, clinical signs are pronounced and, in the
region selected to collect the embryos, prevalence is regularly
monitored. BT was selected mainly because the possibility of
importing infected embryos from BT-endemic regions, such
as that under study, gives rise to genuine concerns among
veterinary authorities in countries which are free of BT,
especially those with large sheep populations. Moreover, the
asymptomatic nature of BT in cattle means that a diagnosis on
clinical grounds alone is usually impossible. VS is another
disease of great concern to veterinary authorities because its
clinical signs are easily confused with those of FMD, but, in
contrast to FMD and BT, the epidemiology and pathogenesis
of VS are poorly understood. Also, VS virus adheres rather
firmly to the bovine ZP and cannot be removed very
effectively by washing.
In the study by Sutmoller and Wrathall ( 3 5 ) , the embryos
were assumed to have originated from a sub-tropical region in
South America where extensive cattle raising, mainly for beef
production, is practised. The region selected for the study is
characterised by a rapid turnover of the cattle population with
large influxes of young cattle for fattening from adjacent
regions. In that region, FMD affects about one herd per
thousand herds a year, while BT and VS viruses appear to
circulate permanendy in the livestock population. Clinical
cases of BT are not observed and VS affects cattle and
members of the equine species only during sporadic
outbreaks (26).
Methodology
Scenario pathway
Figure 2
considered
question is
included).
shows the risk-associated events that were
for the scenario pathway: if the answer to the first
'No', then there is no risk (no infected donor herd
However, if the answer is 'Yes', then the next
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Rev. sci tech. Off. int. Epiz., 16 (1)
Finally, there are the post-collection questions related to
disease observation in the herd of origin and laboratory tests
to detect the presence of the pathogen. A schematic listing of
the above risk-related events is presented in Table I.
Importation of bovine embryos
from an infected region
One or more donor herds infected
with the agent?
In Table I three phases of risk mitigation are presented
according to the three broad lines of defence against the
introduction of diseases by the importation of embryos. The
first phase covers the potential for production of
contaminated embryos and encompasses the disease situation
in the exporting country and/or region of origin, the health
status of the herds and the donor cows from which the
embryos are collected, and the pathogenic characteristics of
the specified disease agent. The second phase covers the risk
mitigation measures which depend on the use of
internationally accepted standards for processing of embryos.
The third phase is the risk reduction resulting from
post-collection surveillance of the donors and donor herds,
and also the possible testing of embryo collection (flushing)
fluids for presence of the disease agent.
Disease in the donor herd detected
by surveillance system?
Disease in the donor herd detected
at embryo collection?
Embryos contaminated by the agent
in the genital tract?
non-exportable viable embryos
Embryos discarded?
Probability of adverse events occurring
exportable viable embryos
Embryos properly washed?
Agent attached to zona pellucida?
Disease detected in donor herds during
storage of embryos?
For each of these risk-associated events (1,2,3, ... n), rather
than a simple Yes' or 'No' answer as implied in Figure 2, there
is a probability P ( l , 2 , 3 , ... n) of conditions that cause the
persistence of the disease agent in the chain of events. The
overall risk that the batch of embryos would contain one or
more contaminated embryos is the product of the
probabilities for each of the events. The evidence on which the
probability estimates were based are all fully documented in
the paper by Sutmoller and Wrathall ( 3 5 ) , but some of the
important points are reiterated here.
Table I
List of risk-associated events for disease transmission by bovine
embryo transfer
Diagnostic tests detect the agent?
Event no. Event
Contaminated embryo
Fig. 2
Risk scenario pathway for possible disease transmission by bovine
embryos destined for international movement
question is 'will the surveillance system and/or the embryo
collection team detect the disease?' If the answer is affirmative
then there is no risk, but if negative, then a risk does exist. The
next consideration relates to whether the pathogen reaches
the embryonic environment. If the answer is Yes', there is a
probability that embryos are contaminated. At this point in
the pathway, embryos are divided in two fractions: f , which is
the fraction of viable exportable embryos, and 1 - f , which is
the fraction of non-exportable viable embryos (Fig. 1). For
fraction 1 - f , the following question is asked: 'Are
non-exportable embryos removed?' For fraction f , two
questions are to be answered: 'Are the embryos properly
washed?' and 'Does the agent adhere or stick to the ZP?'
1.
Inclusion of one or more infected donor herds
First line of defence
2.
Detection of the disease in the donor herd by the animal health
surveillance system
3.
Detection of the disease in the donor herd by the embryo
collection team
4.
Lack of contact between embryos and the disease agent in the
genital tract of a donor
Second line of defence
5.
Removal of the contamination during embryo processing by:
5.1. Detection and removal of embryos which, although viable,
are non-exportable from a disease hazard standpoint
5.2. Proper washing of exportable embryos
5.3. Removal of the disease agent adhering to the zona pellucida
Third line of defence
6.
Detection of the disease in donor herds while the embryos are In
storage
7.
Detection of the disease agent by diagnostic tests on collection
• fluid or other samples
e
e
e
e
Rev. sci tech. Off. int. Epiz., 16(1)
General probability estimates
Certain values used in the simulations are independent of the
disease under consideration while others are not. Examples of
factors which are independent of disease include the number
of donor herds, the fraction of exportable embryos, the
probability of non-exportable embryos remaining in the
exported batch, and the probability of exportable embryos not
being adequately washed. The quantitative estimates for these
values were based on the opinions of several commercial and
non-commercial ET practitioners and were intended to reflect
variable degrees of competence and integrity of the embryo
collection team.
Disease-specific probability estimates
Other values used in the study are disease-specific, for
example: disease incidence, the duration of the disease
episode in the herd and the probability of failure of the animal
surveillance system or the embryo collection team to
recognise the disease before or at the time of embryo
collection.
Prevalence and recognition of clinical signs
The values for the calculated prevalence ( 1 8 ) of FMD and VS
in the area were based on the annual incidence of these
diseases as reported by the Continental Vesicular Disease
Surveillance and Information System ( 6 ) . Based on those
records, for FMD a yearly incidence rate of one infected herd
in 1,000 herds was assumed. Since VS occurred, only
occasionally in the region, an outbreak affecting 19 herds out
of a total of 3 , 9 0 0 was taken as the basis for the study. BT
infection occurs in nearly all of the cattle herds in the area, and
viraemias were assumed to last between 7 and 6 0 days.
During the low-vector season, the likelihood of active
infection was estimated to be lower by a factor of ten.
The probability that the surveillance system or the embryo
collection team fail to detect FMD or VS in donor herds at or
before the time of collection depends on their efficiency. It
would be unlikely that FMD or VS outbreaks would go
undetected, particularly since embryos for export would
usually be collected in well-managed herds with high animal
health standards. However, BT in cattle is usually an
inapparent infection, and clinical expression is rare ( 1 7 , 2 7 ) .
Thus, the probability that infection in the donor herd would
go unobserved by the surveillance system or the embryo
transfer team would be close to 1 0 0 % .
Disease pathogenicity
In the diseases considered, viraemia is a prerequisite for the
contamination of the embryo in the genital tract of the donor
cow. Thus the frequency with which the pathogen would
reach the embryonic environment depends on the likelihood
of
viraemia, which in turn depends on the immune status of
the donor. Most donor farms are likely to maintain a good
FMD vaccination programme and are unlikely to have
recorded this disease in recent years.
231
The prolonged circulation of BT virus in the blood of
BT-infected cattle increases the likelihood of in utero exposure
of the embryo and of contamination of the ZP (39). According
to Bowen et al. ( 8 ) , BT virus would probably be associated
with cellular blood elements. It was assumed, therefore, that
during the high-vector season, about 1 0 % of the cattle in the
herds in the area would be viraemic, but during the
low-vector season this value would be much lower.
Recent studies indicate that viraemia rarely, if ever, occurs
with VS in cattle and pigs (J. House, personal communication;
29). Thus, while low grade transient viraemias could not be
ruled out entirely, the probability of VS virus contamination
of embryos in the genital tract of the donor is likely to be close
to zero. However, in this model a value of 5% was assumed.
Embryo collection and processing
Research has shown that when embryos are washed according
to the procedures described in the IETS Manual, FMD and BT
viruses do not adhere to the ZP ( 3 2 ) . However, in spite of
numerous studies on this aspect, the number of embryos used
was of necessity limited, and on statistical grounds this
obliged the authors to set the range between zero and 1% for
the probability of failure to remove FMD and BT from
contaminated embryos. It appears that VS virus sometimes
adhered to the ZP of embryos which were exposed to VS in
vitro (14, 2 8 , 3 1 ) with 2 0 % to 6 0 % remaining contaminated
following washing. Also, the probability of human error was
recognised. These considerations were reflected in the
probability estimates.
Post-collection observations and laboratory tests
The probabilities that FMD and VS would not be detected in
the donor herds while the embryos are in post-collection
storage were based on the assumption that the importing
country would request post-collection surveillance as part of
the export health certification process. It is unlikely that BT
infection would be detected in cattle herds and for that reason
the probability of failure of the surveillance system would be
close to 100%.
The probability that laboratory tests would fail to detect FMD
infection is remote and the most likely value for false negative
test results was assumed to be about 5%. Also, if tests for BT
virus are to b e used to indicate presence of viraemia, then
testing of blood taken from the donor at the time of embryo
collection should be more sensitive than the testing of
collection fluid. VS virus in collection fluids or blood samples
would, if present, be of very low titre and the testing of such
samples would, therefore, be very inefficient.
Computer simulation
Most probability estimates have a degree of uncertainty, but
when all available evidence is carefully considered it is
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generally possible for the parties concerned to agree on
minimum and maximum probabilities that are reasonably
accurate, and on the most-likely value within this range. The
information is then used to simulate a so-called probability
distribution function (PDF) for each risk event along the
scenario pathway (3, 12). In these studies a computer
program (@PJSK°) was used to simulate PDFs for each of the
P values along the pathway (3, 12) and a total risk PDF to
illustrate the import risk resulting from the complete scenario
pathway, as shown in Figure 3.
12
FMD contaminated embryos is one in 1,000 billion ( 1 0 ~ ) ,
and that risk would be unlikely to exceed one in 100 billion.
At the end of phase 1 the most-likely (mean) risk of a batch of
300 embryos containing at least one FMD-contaminated
embryo is slightly under one in 10 million, and there is only a
5% chance that this will be greater than one in a million. The
second phase would reduce that risk by a factor of 3 0 - 6 0 , and
the third phase by a factor of 4 0 0 - 1 , 0 0 0 .
For BT, during the vector-season, as many as 1 0 % of potential
donors were assumed to be viraemic, and, because it is
difficult to recover embryos without at least some endometrial
trauma, with consequent blood contamination (8) it was also
assumed that most of those would yield BT-contaminated
embryos. According to the simulation results there would be a
chance of 1/10 to 1/20 that the batch of 3 0 0 embryos would
contain at least one BT-contaminated embryo by the end of
the first phase. However, in the low-vector season, with much
less BT infection of herds and individual donors, the
probability of BT-contaminated embryos would be 1/5,000
and this risk is not likely to exceed 1/1,000. The second and
third phases would reduce the risk by factors of 3 0 - 6 0 and
2 0 - 3 0 respectively, and those risk reductions apply to both
the low-vector and high-vector seasons.
A PDF is simulated by carrying out a very large number of
recalculations (iterations), each of which is based on different
random numbers generated by the computer and on the given
distribution parameters such as the minimum, maximum,
and the most-likely probabilities. The results of the
simulations are presented graphically or as statistical reports.
The expected or mean risk is the most-likely outcome of the
simulation and is represented by the midpoint of a PDF. The
other important statistic is the risk value at which 9 5 % of the
iteration results are equal to or below that value.
Simulation results
The simulation results for the contribution made by each line
of defence towards the safety of embryo transfer are shown in
Table II. This Table, together with Figure 3, illustrates the
final import risk of bovine embryos for the three diseases
under consideration. In Figure 3 the graphs in the top row are
the PDFs for FMD, for BT during the high-vector and
low-vector seasons and for VS. The cumulative graphs in the
bottom row show the same data presented differently,
indicating not only the total range of risk, but also the
probability that a risk value will fall within a certain range.
During the high-vector season the most-likely total risk (i.e.
inclusive of phases 1, 2 and 3 ) that a batch of 3 0 0 embryos
would contain contaminated embryos would be 1/50,000,
but this risk would be unlikely to exceed 1/15,000. The most
likely total risk for the low-vector season would be one in 4
million, but would be unlikely to exceed one in a million.
For VS, if embryo collection occurred during a worst case
scenario period (i.e. during an epidemic with 19 of 3,900
herds affected by the disease), the probability that a batch of
300 embryos would contain one or more VS contaminated
embryos would not exceed one in a million by the end of
phase 1. The second and third phases are likely to reduce the
risk by factors of 2-3 and 3 0 - 6 0 , respectively. However, given
For FMD, under the specified conditions (35), i.e. one FMD
infected herd per 1,000 herds per year, the final (mean)
import risk of a batch of 300 embryos contenting one or more
Table II
Risks of disease transmission by embryo transfer in cattle
Phase
1.
2.
3.
Final
r i s k
Foot
Probability of including contaminated embryos
Probability of failure to
remove contamination during
embryo processing
Probability of failure to detect
infected herds post-col lection
Phases 1,2 and 3
and mouth
disease
(a)
1/million
1/10 million
1/30
1/60
(b)
1/400
1/1,000
1/100 billion
1/1,000 billion
a| 95% of the simulated results for the variable are equal or below the value and 5% are higher
b) Mean risk or most-likely risk
Bluetongue
High-vector season
Low-vector season
Vesicular
stomatitis
1/10
1/20
1/30
1/60
1/1,000
1/5,000
1/30
1/60
1/million
1/3 million
1/20
1/30
1/15,000
1/50,000
1/20
1/30
1/million
1/4 million
1/2
1/3
1/30
1/60
1/100 million
1/500 million
10
Low vector season
High vector season
Probability |log values) of contaminated embryos in a batch
of 300 embryos collected under the conditions of the study
Bluetongue
Probability density functions for the risk of foot and mouth disease, bluetongue and vesicular stomatitis by bovine embryo transfer
0
Probability |log, values) of contaminated embryos in a batch
of 300 embryos collected under the conditions of the study
Foot and mouth disease
10
Probability (log valuesi of contaminated embryos in a batch
of 300 embryos collected under the conditions of the study
Vesicular stomatitis
Rev. sci tech. Off. int. Epiz, 16 (1)
233
234
the low probability of contamination of embryos with VS
during the first phase, the risk of one or more embryos
contaminated with VS being present in the imported batch of
300 would be unlikely to exceed one in 100 million.
Rev. sei tech. Off. int Epiz., 16(1)
importing countries stipulate that media must be obtained
from a specified source to avoid this risk. In this study it was
assumed that the media were properly sterilised, and this risk
was not included in the scenario pathway.
Third line of defence
Defence lines and strategic
implications
These QRA studies have confirmed that the risks of disease
transmission by bovine embryo transfer are generally very
low. However, the reasons can vary for different diseases, and
the comparison of FMD, BT and VS serves to illustrate this.
First line of defence
The obvious clinical signs of FMD and VS make it unlikely
that embryos would be collected on an infected farm or from a
diseased donor. However, in the absence of scientific
information that VS viraemia never occurs under natural
conditions, a rather high value had to be assigned to the
probability that VS virus would reach the embryonic
environment. Nevertheless, the first line of defence is almost
as efficient in risk reduction for VS as it is for FMD. This is not
the case with the first line of defence for BT, and especially not
during the vector season when the infection would occur on
nearly all farms in the selected region and a rather high
percentage of animals could be expected to have viraemia. In
addition, due to the asymptomatic nature of BT, there is a
high probability of failure to detect this infection. Of course,
embryo collection during the low-vector season would gready
reduce the risk of BT transmission.
In these studies the authors did not consider the potential
risks arising from an embryo collection team moving from
one farm to another, or using contaminated media for embryo
collection and washing. Concerns about these are justified,
but an assumption was made that the sanitary conditions
demanded by the importing country would prevent these
risks from occurring.
Second line of defence
Risk-associated factors to be addressed at the second line of
defence include detection and removal of non-exportable
embryos and adequate washing of exportable embryos.
Human failure in these respects is the predominant risk here,
and an effective system for accreditation of embryo collection
teams will greatly help to ensure their integrity and technical
ability. The second line of defence is basically the same for
FMD and BT, since both agents can be efficiently removed
from the ZP by washing, but it is less effective for VS which
has a tendency to adhere to the ZP. The risk that disease
transmission might occur as a result of using contaminated
media for the collection, processing or storing of embryos is
very important and should not be overlooked. Some
The effectiveness of the third line of defence depends initially
on the ease of recognising the disease in the herd of origin
while the embryos are in post-collection storage. Clinical signs
are not missed easily in cases of FMD and VS, but this is not
the case for BT. Laboratory tests could be employed to detect
FMD virus in embryo collection fluid, but this would require
special bio-containment laboratory facilities. For BT the
testing for viraemia is more likely to be effective than the
testing of collection fluid. Laboratory tests for VS were not
considered effective.
Risk of disease establishment in
the importing country
For
catastrophic contagious diseases, most importing
countries will wish to take account only of the import risk.
However, for other diseases, such as those which are
transmitted exclusively by vectors, further scenarios might
also be considered, including the minimum infective dose of
the pathogen and the availability of a competent vector in the
importing country. With regard to FMD, import authorities
tend to be interested primarily in preventing the importation
risk and less in the risk of disease establishment, whereas for
diseases such as BT, the risk of establishment may also be
considered. BT infection is seasonal, non-contagious and
vector-bome, and disease spread seems to be limited by
ecological boundaries. The regional nature of BT is evident
from studies conducted in the Okanagan Valley of British
Columbia (Canada), and in parts of the USA and Australia
(17, 30). If the vector does not occur in the receiving country
the recipient may still become infected, but since the disease is
not contagious it will not become established. Such ecological
factors can be taken advantage of to give further reduction in
risk. For example:
- embryos can be collected during the period when the
lowest number of viraemic cattle would be expected, for
instance, from 100 days after the end of the vector season
until the beginning of the next season (30).
- embryos can be transferred in the receiving region at the
beginning of the period with the lowest vector activity, thus
reducing the risk of establishment of BT in the receiving
region.
While the pathogenesis of VS is not well understood,
geographical distribution of the disease suggests that the risk
of establishment outside certain ecological boundaries is very
low. Ecological factors seem to be related to types of pastures,
235
Rev. sci tech. Off. int. Epiz., 16(1)
ambient temperatures and amounts of rainfall. For reasons
unknown, certain areas and farms are prone to experience VS
at frequent intervals. Under those circumstances it appears
that the probability of the establishment of VS outside such
areas is virtually zero.
Conclusions
Risk assessment is a valid approach for estimadng and
managing the risks of embryo importation and provides
veterinary authorities with an alternative methodology to the
disease categorisation system of the IETS (5) on which to base
their import/export decisions. The IETS categorisation system
gives no quantitative guidance on the meaning of the term
'negligible risk' and is based solely on research data from
studies on embryo-pathogen interactions in vitro and in vivo.
For a QRA, however, in addition to research data on
embryo-pathogen interactions, one can consider disease
prevalence, effectiveness of Veterinary Services and
competence of the embryo collection team, as well as the
epidemiology and pathogenesis of the diseases concerned.
The QRA results of this study show that the latter factors play
a major role in determining the risk of disease transmission by
embryos. For instance, although FMD and BT were both
placed by the IETS in Category 1 ('negligible risk') because
research had shown convincingly that the agents of both
diseases can be removed effectively from the ZP by washing,
the estimate of risk for BT made here is larger than for FMD
because of the asymptomatic nature of BT in cattle. The fact
that VS is listed in IETS Category 4 ('Diseases or disease agents
on which preliminary work has been conducted or is in
progress') may lead to undue concerns among regulatory
officials, and unnecessary trade restrictions. According to
estimates made here, VS has an exceedingly small probability
of being transmitted by international embryo movement
because its presence, like that of FMD, is easily detected on
the farm by clinical surveillance.
Acceptably low risks of transmitting disease through the
international movement of bovine embryos depend initially
on a low disease incidence in the exporting region and on
disease signs which can easily be recognised by those involved
in disease surveillance. Nevertheless, embryo transfer
practitioners must be aware that estimates of their
competence (e.g. in embryo washing and in excluding
embryos with non-intact ZPs) and their integrity (e.g. in
embryo certification) also have substantial effects on embryo
import risks, and estimates of these are incorporated into
mathematical models and equations from which the risks are
quantified. Thus, in commending the QRA approach to
veterinary authorities for the evaluation of embryo transfer
risk, the importance of both a reliable disease surveillance
system in the exporting region and the integrity and
competence of embryo collection teams is emphasised.
The use of the QRA methodology is basically simple and
when new data become available the results can be easily
re-worked. A QRA can also indicate the areas in which
additional research information would be most useful. For
instance, if new research on embryo-pathogen interaction in
vitro is proposed in the hope of enabling a more precise
appraisal of IETS washing procedures, such studies would
only be justified if the QRA completed beforehand indicated
that the results might significanrly reduce the total import
risk. In addition, it should be noted that QRA is a valuable
tool for assessing the safety of the movement of embryos of
species for which little or no research data are available on
embryo-pathogen interactions.
Acknowledgements
The authors are both members of the Import/Export
Committee of the International Embryo Transfer Society
(IETS) and much of the content of this paper is based on
discussions and work associated with that Committee. Several
members of the Committee have also reviewed the paper and
the authors acknowledge their valuable contributions and
suggestions.
236
Rev. sci tech. Off. int. Epiz., 16 (1)
Risques de transmission de maladies lors des transferts
d'embryons bovins
P. Sutmoller & A.E. Wrathall
Résumé
Les principes directeurs garantissant l'innocuité des transferts internationaux
d'embryons bovins sont énoncés dans le Code zoosanitaire international de
l'Office international des épizooties ; par ailleurs, les recommandations relatives à
la manipulation des embryons sont décrites dans le Manuel de la Société
internationale de transfert d'embryons, en tenant compte des nombreux travaux
sur l'interaction embryon-agent pathogène. L'évaluation des risques s'inscrit
dans la logique de ces textes, dans la mesure où elle fournit aux Administrations
vétérinaires un ensemble complet d'informations sur lesquelles elles peuvent
fonder leurs décisions d'importation/exportation. L'évaluation des risques inclut
l'estimation de la prévalence des maladies, de l'efficacité des Services
vétérinaires et de la compétence de l'équipe de collecte des embryons. Elle
prend également en compte l'épidémiologie et la pathogénie de la maladie
considérée.
Les auteurs illustrent l'application de l'évaluation des risques lors des transferts
d'embryons par une étude comparative des probabilités de transmission de la
fièvre aphteuse, de la fièvre catarrhale du mouton et de la stomatite vésiculeuse
par les embryons bovins. L'évolution du scénario du risque a été divisée en trois
phases. La première concerne la probabilité d'infection des embryons, laquelle
dépend de la situation sanitaire de la région exportatrice, du statut sanitaire des
femelles donneuses et des cheptels d'origine et, enfin, des caractéristiques
pathogènes de l'agent causal. La seconde phase a trait aux méthodes
d'atténuation du risque, notamment au respect des normes, agréées à l'échelle
internationale, pour la manipulation des embryons. Quant à la troisième phase,
elle analyse les moyens de réduction du risque, à savoir la surveillance, après
collecte, des femelles donneuses et des cheptels d'origine et la recherche de
l'agent pathogène dans les liquides de collecte (ou de lavage) des embryons.
Il ressort de cette évaluation que le risque de transmission de maladies lors de
transfert international d'embryons bovins peut être considéré comme faible
lorsque l'incidence de la maladie dans la région exportatrice est elle-même faible
et lorsque les symptômes de l'infection sont aisément identifiables. La
compétence de l'équipe chargée de manipuler les embryons revêt également la
plus haute importance ; dans le cas de la fièvre catarrhale du mouton, l'écologie
du vecteur joue un rôle majeur.
L'évaluation des risques offre non seulement une base rationnelle aux décisions
d'importation/exportation, mais elle sert également à évaluer les résultats de
nouvelles recherches et à apprécier les garanties sanitaires entourant les
transferts d'embryons d'autres espèces, pour lesquelles aucun travail de
recherche ou presque n'a été réalisé en matière d'interactions embryon-agent
pathogène.
Mots-clés
Bovins - Embryons - Évaluation des risques - Importation - Transmission des maladies.
237
Rev. sci tech. Off. int. Epiz., 16 (1)
Riesgos de transmisión de enfermedades mediante transferencia
de embriones bovinos
P. Sutmoller & A.E. Wrathall
Resumen
El Código zoosanitario internacional de la Oficina Internacional de Epizootias
contiene las directrices que deben aplicarse para garantizar la inocuidad de los
intercambios internacionales de embriones de animales de granja. El Manual de
la Sociedad Internacional de Transferencia de Embriones, por otra parte, ofrece
recomendaciones acerca del procesamientos de los embriones, basadas en
numerosas investigaciones sobre la interacción entre los embriones y los
agentes patógenos. La evaluación de riesgos constituye la continuación lógica de
estos textos, dado que proporciona a las Administraciones veterinarias un
conjunto completo de información sobre la cual basar sus decisiones en materia
de importación/exportación. Evaluar los riesgos significa evaluar la prevalencia
de la enfermedad, la efectividad de los Servicios Veterinarios y la aptitud del
equipo encargado de la recolección de los embriones. El proceso de evaluación
debe tomar asimismo en cuenta la epidemiología y patogenia de la enfermedad
en cuestión.
Los autores ilustran la aplicación de la evaluación de riesgos al movimiento de
embriones mediante la comparación de las probabilidades de que embriones
bovinos transmitan la fiebre aftosa, la lengua azul y la estomatitis vesicular. El
procedimiento de análisis de las diferentes situaciones de riesgo fue dividido en
tres etapas. La primera fase considera la probabilidad de que el embrión
contraiga la infección, factor que depende tanto de la situación de la enfermedad
en la región exportadora como del estado sanitario de las hembras donantes y
rebaños de origen y de las propiedades patógenas del agente infeccioso. La
segunda fase estima la atenuación del riesgo que se sigue de la aplicación de
normas internacionalmente aceptadas para el procesamiento de los embriones.
La tercera, por último, evalúa la reducción del riesgo resultante de la vigilancia de
las hembras donantes y rebaños de origen después de la recolección y de las
pruebas de detección de agentes patógenos aplicadas a los líquidos de
recolección (y lavado).
Esta evaluación puso de manifiesto que la consecución de niveles bajos de riesgo
de transmisión de enfermedades debido a los intercambios internacionales de
embriones bovinos depende, en primera instancia, de una baja tasa de incidencia
de la enfermedad en la región exportadora y de la existencia de síntomas de
enfermedad fácilmente reconocibles. Un procesamiento adecuado del embrión
es asimismo de la mayor importancia, al igual que, en el caso de la lengua azul, las
características ecológicas del vector.
Además de proporcionar una base lógica para la toma de decisiones en materia
de importación/exportación, la evaluación de riesgos permite valorar el posible
resultado de nuevas investigaciones y estimar el nivel de seguridad que ofrece el
movimiento de embriones de especies sobre las cuales existe poca o ninguna
información relativa a las interacciones entre embriones y patógenos.
Palabras clave
Embriones - Evaluación de riesgos - Ganado vacuno - Importación - Transmisión de
enfermedades.
238
Rev. sci tech. Off. int. Epiz., 16 (1)
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