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Rev. sci. tech. Off. int. Epiz., 2012, 31 (3), 761-775

A quantitative assessment of the risk

of introducing foot and mouth disease virus intothe United States via cloned bovine embryosB. 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]

SummaryThe 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 theUnited States (USA) is that it might enter via cloned embryos. A quantitative riskassessment model was developed to determine the scenarios (withmathematical probabilities) that could lead to the introduction and maintenanceof FMDV via the importation of cloned bovine embryos. Using @RISK softwarewith Monte Carlo simulation involving 50,000 iterations, the probability ofintroducing FMDV via cloned embryos was estimated to be 3.1 107. Given thecurrent cloning protocol, and assuming the annual importation of 250 to1,700 (mean = 520) cloned embryos, the expected number of infected embryosranges from 1.1 107to 4.4 103 (mean = 1.6 104) per year. Critical pathway

analysis showed that the risk of FMDV entering the USA by this route isextremely low.

KeywordsCloning Critical pathway analysis Foot and mouth disease Monte Carlo simulation Quantitative risk assessment United States.

IntroductionCloning by somatic cell nuclear transfer (SCNT) is arelatively new technique in which animals are producedasexually by taking a differentiated somatic cell and fusingit with, or microinjecting it into, an enucleated oocyte. Thedonor cells are commonly obtained by biopsy fromselected adults (26, 41), of which there is an unlimitedsupply (9). However, the cytoplast of a mature oocyte is

the only type of cell identified as having the capacity toreprogramme the donor cell nucleus correctly to expressthe genes required for early development (30).

Somatic cell nuclear transfer continues to be developedand improved, but as yet there is no method that isuniversally employed. The basic steps (Fig. 1) are commonto most SCNT procedures, but it is a complex, technicallydemanding and inefficient process (10, 21, 27, 32).Nevertheless, it is a promising technique for preserving andpropagating superior genes with known performance traits(4, 10). This is of particular value in food animalproduction because it may take years to prove the merit of

a sire or dam. Cloning by SCNT has been used successfullyto produce livestock of various species, including thecamel (40).

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762 Rev. sci. tech. Off. int. Epiz., 31 (3)

Fig. 1Main steps of somatic cell nuclear transferSource: European Food Safety Authority, 2008 (8)

Oocyte source

Genitor

Sexual reproduction

Clone offspring (F1)

Removal of nucleus

and polar bodyfrom oocyte

Cultured somatic cells(often fibroblast)

Cell injection

Embryo clone

Surrogate dam

Clone (F0)

Electrofusion and activation

I

I

H

G

F

B

D C

E

A

Nucleus source

B

from oocyteand polar body

Removal of nucleusOocyte source

A

Nucleus source

Cultured somatic cells

Nucleus source

E

D

F

E

D

Cell injection

C (often fibroblast)Cultured somatic cells

G

H


Embryo clone


Genitor

I

Surrogate dam

I

Genitor


Sexual reproduction

Clone (F0)


A: cell donorB: oocyte donorC: cultured cellsD: enucleation

E: cytoplastF: injection of somatic cell into cytoplastG: electrofusion of oocyte and cell membraneH: embryo clone

I: embryo transfer into a surrogate dam generatingclone (F0); F1 generated by mating of the clone (F0)with a normal partner

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NO

RISK

Development of the scenario pathway

The authors applied epidemiological principles to developa 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 thepurpose of the risk assessment, these events are termednodes) (Nodes 1 to 10), was developed. From this theauthors determined the conditions (with associatedstatistical probabilities) that could lead to the introductionand maintenance of FMDV in the cloning pathway. Theparameters for the occurrence of the FMDV infection eventat each node, represented by distribution functions tocapture variability, were estimated using publishedliterature (1, 2, 36). Based on the ranges of estimates,

Rev. sci. tech. Off. int. Epiz., 31 (3)764

various distribution functions (including Uniform, Pert,and Beta) were used to calculate expected values at eachnode using @Risk software (Version 5.7, PalisadeCorporation). The sum of the product of the probability

distributions P1P4 and P5P7, multiplied by P8P10(Table I), was considered to be the overall probability ofintroducing FMDV into the USA via cloned embryos. Inthe analyses, Monte Carlo simulations (a computerisedmathematical technique for iteratively evaluating adeterministic model using sets of random values as inputs)were set at 50,000 iterations. For any factor that hasinherent uncertainty, Monte Carlo simulation buildsmodels of possible outcomes by substituting valuesrandomly from a range of possible values (the probabilitydistribution). It then calculates the results repeatedly(iterations), each time using a different set of random

C-O-C: cumulusoocyte complex

FMDV: foot and mouth disease virusFig. 2A scenario tree for the likelihood of introduction of foot and mouth disease virus into the USA via cloned embryos

INITIATING EVENT: Importation of cloned embryos

N

N N

Y Y

N

YY

N

Y

Y

Y Y

N

Y

N

N

N

Y

N

Infected cloned embryos imported into the USA

Is selected somatic cell donor infected?P1 NODE 5

NODE 6

NODE 7

NODE 2

NODE 1

NODE 3

NODE 4

NODE 8

NODE 9

NODE 10

Can diagnostic tests fail to detectFMDV-infected somatic cell donor?

P2

Can the tissue culture remove FMDV from donor cells?P3

Is slaughtered oocyte donor infected?P5

Is the oocyte/C-O-C infected?P6

Can the maturation and washing processesremove FMDV from the oocyte?

P7

Can diagnostic tests fail to detectFMDV in tissue culture?

P4

Can the washing process removeFMDV from the developing embryo?

P8

Can FMDV-infected embryos developinto exportable blastocysts?

P9

Can FMDV infection be detectedin exportable blastocysts?

P10

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Rev. sci. tech. Off. int. Epiz., 31 (3) 765

values from the probability functions at each node. Theanalysis range shows all the possible outcomes, such as theones suitable for the most conservative, or intermediate-level, decisions. This quantitative risk assessment (QRA)model enables the regulatory authorities to compare theacceptable level of risk with the likelihood of occurrence ofa scenario.

Estimation of the foot and mouth disease virusrisk 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 vesiculardisease 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 theMiddle and Far East, is characterised by fever, loss ofappetite, salivation and vesicular eruptions on the feet,mouth, and teats (39). Morbidity can reach 100% in

susceptible animal populations (2), although in vaccinatedpopulations only about 5%10% of affected cattle herdsmanifest the disease (35). This is the case in regions such

as South America, where vaccination often covers about90% of each herd (36).

Pedigree animals, such as those that might produce thedonor cells that the cloning procedure is designed to

propagate, are normally subject to rigorous healthmonitoring and surveillance during their lifetime (8, 45).However, the clinical diagnosis of FMD can sometimes bedifficult, depending on the virulence of the virus andvaccination status. The fact that the acute stage of infectionmay be followed by prolonged, symptomless, persistentinfection (the carrier state) further complicates thesituation (2, 36). The proportion of such carriers dependson the incidence of the disease (or infection) and theimmune status of the population, but carriers are foundrelatively 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 authorsquoted a carrier state of 50% in Brazil after a vaccinebreakdown. The proportion of animals that becomecarriers under experimental conditions is variable butaverages around 50% (2, 36).

Based on the above evidence, the probability that a donorof somatic cells is infected is estimated to equal theproportion of persistently infected animals in enzooticareas, 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 to3.5 years post infection. Carriers have also been shown tomount cell-mediated and humoral immune responses andto secrete virus-specific IgA in saliva (reviewed by Kumar[17]). The gold standard test for FMD antibody detectionis the virus neutralisation test (VNT). However, when largenumbers of sera need to be tested, screening is usuallydone by enzyme-linked immunosorbent assay (ELISA).

A solid-phase blocking ELISA for the detection ofantibodies directed against type O FMDV showed asensitivity of 99% (relative to the VNT) when used to test148 positive cattle, goat and sheep sera collected fromFMDV-infected Dutch farms. The ELISA also correctlyscored 398 of 409 (97.3%) experimentally derived positivesera (6). The Danish C-ELISA, which had a reported 92%(35/38) sensitivity based on experimental infection, washowever only 71% sensitive at the recommended cut-offwhen used to test samples from naturally infected cattle(5).

Over the years, several ELISA techniques, mainly targetingnon-structural viral proteins (NSP), have been developed.The initial estimates of the analytical sensitivity needed todetect baculovirus-expressed FMDV non-structural

proteins 3AB and 3ABC were 73% (22/30) and 90%(27/30), respectively. The 3AB and 3ABC antigens used inthis particular ELISA were also able to differentiate

Table ISummary 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*P10C-O-C: cumulusoocyte complexFMDV: foot and mouth disease virus

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FMDV-infected pigs from vaccinated pigs (7). Kumar (17)determined the sensitivity of a 3A-NSP-ELISA by testing382 known positive sera; only three of these sera gave anegative reaction (99.2% sensitivity). Nowadays, more

sensitive assays such as real-time polymerase chainreaction (PCR) (97% sensitivity) have been developed todetect the presence of viral RNA for all seven serotypes ofreference FMD viruses (17).

Based on the above evidence, the probability that screeningand confirmatory tests would fail to detect FMDV in theinfected donor of somatic cells is estimated to range from0.01 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-RS-2) cells, baby hamster kidney (BHK-21) cells, Chinesehamster ovary (CHO) cells, human mammary glandepithelial cells, lamb kidney cells, MadinDarby bovinekidney (MDBK) cells and bovine thyroid (BTY) cells,support the growth of FMDV (14, 17, 18, 28, 29). Giventhat the virus is cytocidal, infected cells exhibitmorphological alterations, commonly called cytopathiceffects, which include cell rounding and alteration andredistribution of internal cellular membranes (12). The BTYcells show the best sensitivity for FMD virus detection (3).

According to Khawaja et al. (16), 42 of 62 known virus-positive 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 canalso be assayed by ELISA (1, 16), fluorescent antibodyreaction (25), animal inoculation (24) or PCR (17, 23). Ingeneral, 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 cellcultures, animal inoculation, serology and PCR would fail todetect FMDV in persistently infected somatic cells isestimated to range from 0.02 to 0.2 (mostlikely: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 tocells in cell culture requires specific receptors on the cellsurface. Accordingly, modification of these receptors caninhibit attachment of the virus to cells. In this regard,

Jackson et al. (14) found that including heparin in the cellculture completely inhibited virus attachment to fixedcells; even attached viruses could be eluted by heparin.

Wild & Brown (43) had previously indicated that trypsin-inactivated FMDV failed to adsorb to pig kidney tissue

culture cells held in suspension, which suggests thattrypsin is able to remove or block a specific attachment siteon the protein coat of the virus.

Similarly, ODonnell et al. (28) exposed human mammarygland epithelial cells to FMDV in the presence ofchlorpromazine, which inhibits clathrin-mediatedendocytosis, and assessed the number of virus-positive

cells after 4 h. They determined that there were about50% FMDV-infected cells per field in the control culturescompared with about 4% in the chlorpromazine-treatedcultures. La Torre et al. (18) treated monolayers ofBHK-21 cells lytically infected with FMDV, or lineC1-BHK-Rcl persistently infected with FMDV, withincreasing concentrations of ribavirin in the culturemedium. The ribavirin concentration that reduced thevirus yield to 50%, 2448 h after addition of the drug, was30 g/ml to 50 g/ml for the lytic infection and 3 g/ml to6 g/ml for the persistent infection. Concentrations ofribavirin of 150 g/ml or higher resulted in a decrease invirus production, and reduced infectious intracellular

RNA below detectable levels. Given that no renewedFMDV production occurred during subsequent serialpassages, the authors concluded that treatment withribavirin had removed the FMDV from the persistentlyinfected Cl-BHK-Rcl cells. They also emphasised thatseveral of these ribavirin-cured cell lines have been derived,passaged, frozen, thawed, and regrown by the sameprocedures used for BHK-21 cells, without detectable lossof viability.

Based on the above evidence, the probability that a somaticcell culture line would still be infected after its treatment

during tissue culture with FMDV-inhibiting drugs isestimated 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 byAlexandersen et al. (1), 15%20% of cattle and sheep inAsiatic Turkey were found to be carriers. The same reviewquoted a carrier state of 50% in Brazil after a vaccinebreakdown. The proportion of exposed animals thatbecome carriers under experimental conditions is variablebut averages around 50% (2, 36).

Based on the above evidence, the probability that donors ofthe oocytes are infected is estimated to equal theproportion of persistent infection in enzootic areas,i.e. 0.15 to 0.5 (most likely: 0.20)______________________P5.

Node 6. Could the cumulusoocyte

complexes and the oocyte be infected?Infection with FMDV is initially established in the non-cornified epithelium of the pharyngeal area and thenspreads to regional lymph nodes and via the bloodstreamto cornified epithelial cells of the skin and mouth. Allsecretions and excretions, including saliva, nasal and

lachrymal fluids, milk and expired breath, becomeinfectious. 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). Thevirus is highly concentrated in the follicular fluid andovarian tissues, although the ovaries of convalescent cowskilled six weeks after being diagnosed with the disease

were not infectious (22), signifying that FMDV does notpersist for long in the ovaries of infected cows.

Based on the above evidence, the probability that C-O-Cscould be contaminated in the reproductive tract of females(persistently) infected with FMDV is estimated to rangefrom 0 to 0.27_________________________________________ P6.

Node 7. Would the processes of in vitro maturationand washing remove foot and mouth disease virus

from the oocyte/cumulusoocyte complexes?Mebus & Singh (24) used in vivo uterine flushing to collecta total of 436 embryos and unfertilised ova from

FMD viraemic cows. After appropriate washing (accordingto the International Embryo Transfer Society [IETS]protocols), degenerated embryos (n = 144) and unfertilisedova (n = 64) were assayed either in cell culture or byinoculating them intradermally into the tongues of steers.None of the sonicated ova and embryos was found to beassociated with the virus. Similarly, Jooste (15) collectedC-O-Cs from the ovaries of slaughtered cows and exposedthem to FMDV by adding the virus to the maturationmedium. Although the FMDV-exposed C-O-Cs andcumulus cells produced cytopathic effects on pig kidneycells even after washing, oocytes that had been denuded of

cumulus by treatment with morpholino-ethanosulphate(MES) were negative for the virus when tested by cellculture and by PCR.

Based on the above evidence, the probability that in vitro-cultured, denuded, washed, and treated oocytes would carryFMDV 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 thevirus in their reproductive tracts (24), the intact ZP,

especially that of in vivo-derived embryos, acts as a barrierfor the majority of infectious agents, including FMDV (36).It has also been shown that FMDV does not adhere firmlyto the intact ZP of in vivo-derived embryos (31, 38),provided that the latter are properly washed according toIETS protocols (34). To investigate whether FMDVinteracts differently with bovine embryos produced in vitro,a suspension of FMDV was added to batches of seven-day-old in vitro-fertilised embryos which were then incubatedwith virus for periods of 1 h, 2 h or 4 h. After the embryoshad been washed ten times, they were pooled and groundup to form a suspension, and this suspension was then

assayed on cell cultures for FMDV. Assays for FMDV werealso conducted on the first and second wash and on thepooled sample constituting the eighth, ninth and tenth

washes by cell culture and PCR. Although almost allsamples of the first and second wash, and both developedand degenerated embryos (8 out of 12 batches), producedFMDV cytopathic effects, all pooled eighth to tenth washes

were cytopathogenically negative (23).Similarly, Singh et al. (31) exposed 169 in vivo-derivedZP-intact bovine embryos to 106 plaque-forming unitsper/ml of FMDV. After standard washing, no infectiousvirus was detected on any of the embryos. However, FMDVinfectivity was found in association with 14 of 42 hatched(ZP-free) bovine embryos and four of the 124 ZP-intactporcine embryos. It was noted that the level of infectivitydid not increase with further incubation, which suggeststhat replication of the virus in embryonic cells is unlikely.In a later study (24), a total of 653 in vivo-derived bovineembryos/ova were exposed to FMDV in vitro or by

collecting them from FMDV-inoculated animals. Theseembryos/ova were washed and then tested for FMDVinfectivity. The virus was not found to be associated withany of the embryos/ova. Any residual infection could beremoved by adding trypsin to the embryo wash fluids; thisinactivates FMDV by removing or blocking a specificattachment site on the protein coat of the virus (43).

Based on the above evidence, the probability that embryoclones could still carry FMDV after proper washing andtrypsin treatment is estimated to range from 0.01 to0.08 (most likely: 0.02) _______________________________ P8.

Node 9. Would embryos infected with foot and mouthdisease 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 FMDVin vitro reached blastocyst stage, compared with 22.2%(55/250) for the non-exposed control embryos. Usingtypical culture conditions, it was found that 105 of569 fused couplets (18.5%) developed to bovine clonedblastocyst stage (46).

Based on the above evidence, the probability that FMDV-infected 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 fluidsis usually made by detection of specific FMDV antigensusing ELISA (15), cell culture (22, 23, 24, 31), animalinoculation (22, 24, 31), or PCR (15, 23), and thesensitivity of these techniques is probably reasonablycomparable with their sensitivity in tissue samples. In onestudy (23), 8 of 12 batches of first and second wash fluid

samples from experimentally contaminated embryocultures produced cytopathic effects. In general, thesensitivity of these tests ranges from 70%80% in cell


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culture (16), 70%90% in ELISA (1) and 70%97% inPCR (17).

Based on the above evidence, the probability that

diagnostic tests would fail to detect FMDV-infectedembryos or FMDV-contaminated wash fluids is estimatedto range from 0.03 to 0.10 ___________________________P10.

Effect of the numberof imported embryos on the estimated risk

Most in vitro systems use oocytes collected either from theovaries of slaughtered cows or by ovum pick up (OPU).On average, it is possible to recover up to 12 good qualityoocytes from a slaughtered cow (15). Using OPU, on theother hand, Yang et al. (46) obtained 8.2 C-O-Cs per

heifer. The maturation and fusion rates of bovine oocyteswere 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) developmentto blastocyst stage. According to the same study, 4 of18 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, usingadult fibroblast-like cells as the donor cells for theproduction of cloned embryos. On the other hand, Wells

(41) reported a pregnancy rate of about 65%, althoughonly 13% (n = 988) of transferred cloned embryos resultedin calves delivered at full term.

Assumptions made

Although the systems used for the production of clonedembryos are very diverse, procedures for managing animalhealth risks associated with cloned embryos have beendescribed (45). However, specific information regardingFMDV transmission through cloned embryo productionand transfer is lacking. For this study, therefore, relevant

information regarding the possible interaction of FMDVwith cloned embryos had to be extrapolated, mainly fromstudies conducted on the interactions of FMDV and otherpathogens with bovine oocytes and embryos. In particular,the research focused on the potential for embryocontamination either from somatic cell or oocyte donors.

As a valuable pedigree animal, a donor of somatic cells willalmost certainly have been subjected to health monitoringand surveillance (8, 45). Oocytes from which the recipientcytoplast is produced are, on the other hand, collectedfrom the ovaries of randomly selected females slaughteredat commercial slaughterhouses (15). Given that there are

no specific new risks identified with SCNT cloning (45),the primary animal health risks are associated with thehealth status of the animal from which the ovaries are

harvested. For this reason, the potential risks (and therespective risk mitigations) for the somatic cells andoocytes have been dealt with separately, prior to theirfusion to produce the cloned couplet. The potential risk

after the fusion has been assessed by reference tointernationally accepted protocols for processing embryos(34) and to the results of testing degenerated embryos andwash fluids for FMDV. The risk of introduction ofextraneous infectious agents such as FMDV, fromsupporting cells and animal products such as serum, wasnot addressed in this study. It is assumed that the cloningindustry will have observed the internationally agreedrecommendations to ensure that media, reagents andequipment, as well as the working environment, are free ofpathogens and contaminating microorganisms (33).

Whether this assumption is justified is difficult toascertain, but it should be judged by the regulatory

authorities in the USA.

The number of imported cloned embryos was initiallysuggested to be 100 per year. This assumption takes intoaccount the number of companies engaged in cloning andvarious applications of cattle cloning, which includeimproving disease resistance (41). Given that the success ofcloning is measured by having a live offspring (and asurviving dam), the efficiency of the cloning procedure iscritical in determining the number of imported clonedembryos. Based on these facts, and in order to provide arealistic risk estimate, a yield of between 10 and

50 (mean = 30) cloned offspring per year was assumed.The number of imported cloned embryos, which rangesfrom 250 to 1,700 (mean = 520), was thus obtained bymodelling contingent on the range of clones produced peryear, rather than using a fixed number of imported clonedembryos as originally suggested (details are provided inTable II).

ResultsIn this study, critical pathway analysis was applied toevaluate the risk of introducing FMDV into the USA viacloned bovine embryos created in endemically infectedregions outside the USA. Specific inputs for the model andrespective calculations are displayed in Tables I and II. Asummary of the results is displayed in Table III. The riskestimate (3.1 107) and the probability density/cumulative probability distribution are shown in Fig. 3 fora better graphical presentation. Assuming the importationof 250 to 1,700 cloned embryos per year, contingent onproducing 10 to 50 cloned offspring, and using the datagiven in Tables I and II as inputs, the expected number of

FMDV-infected cloned embryos imported into the USA peryear is 1.6 104 (Fig. 4). Correspondingly, Fig. 5 depictsthe number of years (5.5 104) it would take before an


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FMDV-infected cloned embryo is imported into the USA(details are provided in Table III). According to thesensitivity analysis (Fig. 6), the inputs showing the highestvariability/uncertainty were: the FMDV status of the C-O-Cs, the effectiveness of the embryo washing protocol andembryo testing procedures, and the FMDV status of oocytedonors.

DiscussionThe infectious hazards posed by cross-border movement ofcloned embryos to the livestock industry, and in somecases to human health in the importing country, have notbeen well elucidated. This makes regulatory authoritiesuncomfortable with the international trade of clonedembryos between nations. Nevertheless, QRA is a logicalapproach that could provide these authorities withplausible information on which to base their decisions. Inthis study, the authors applied QRA methodology toevaluate the risk of introducing FMDV into the USA viacloned embryos imported from regions where FMD isendemic. As shown in the results section, the risk is verysmall, less than 3.1 107. This is mainly due to thebeneficial effect of embryo washing procedures and the

efficiency of diagnostic tests. Oocyte/embryo washing,especially if combined with the use of trypsin, can easily beincorporated into SCNT techniques and has been shown tolimit FMDV transmission effectively when conventionallyproduced embryos, either exposed to the infectious agentin vitro or collected from infected donors, were transferredinto disease-free recipients (33). In fact, digestion of thesource tissues with trypsin is a routine procedure in tissueculture (25) and cattle cloning (9, 10, 42). Trypsin isknown to inactivate viral particles attached to the ZP(33, 45). The assessment therefore substantiates earlier


Table IISummary of parameters for estimating the number oftransferable embryos

Probability Description Distribution

A No. of oocytes per cow/heifer RiskUniform (8, 12)

B Maturation rate of bovine oocyte RiskBeta

(1100+1,(13571100)+1)

C Fusion rate of mature oocyte RiskBeta (569+1,(906569)+1)

D Cleavage rate of fused oocyte RiskBeta (300+1,(569300)+1)

E Blastocyst development rate RiskBeta (105+1,(300105)+1)

of cleaved oocyte

F Pregnancy rate of embryo RiskPert (13.0%, 22.2%, 65.0%)

G Pregnancy yielding live calf (clone) RiskUniform (25.0%, 30.0%)

H Live clones produced per year RiskPert (10, 30, 50)

Number of embryos imported per year = [1/(F*G)]*H

Table IIIThe results of 50,000 iterations showing: 1) probabilities at each node, and 2) the probability of introducing foot and mouth diseasevirus into the USA, the number of cloned embryos imported per year, the number of infected embryos imported per year, and thenumber 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 1.5 101 1.6 101 2.4 101 3.5 101 4.8 101

P2 1.1 102 1.7 102 7.0 102 1.6 101 2.8 101

P3 2.4 105 1.0 102 1.1 101 2.7 101 4.7 101

P4 2.0 102 2.0 102 6.0 102 1.1 101 1.8 101

P5 1.5 101 1.7 101 3.3 101 4.8 101 5.0 101

P6 1.9 106 1.0 102 1.4 101 2.6 101 2.7 101

P7 2.9 107 2.5 103 2.5 102 4.8 102 5.0 102

P8 1.0 102 1.6 102 3.5 102 5.8 102 7.8 102

P9 1.2 101 1.2 101 1.3 101 1.4 101 1.4 101

P10 2.0 102 2.4 102 5.7 102 1.1 101 1.8 101

Probability that an 3.8 1010 1.7 108 3.1 107 1.0 106 4.6 106

imported embryois contaminated

No. of cloned embryos* 2.5 102 2.7 102 5.2 102 9.6 102 1.7 103

imported per year

No. of infected embryos 1.1 107 7.3 106 1.6 104 5.6 104 4.4 103

per year

Years until infected 5.3 102 3.1 103 5.5 104 2.0 105 6.9 106embryo is imported

*Contingent on the assumption of producing 10 to 50 cloned offspring per year

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Fig. 4Probability density function for the number of embryos infected with foot and mouth disease virus imported into the USA per year

Fig. 6Regression sensitivity analysis for the likelihood of introduction of foot and mouth disease virus into the USA via cloned embryos

0.02

5% 90% 5%

0.0.00050.0010.00150.0020.0025

0.0030.00350.0040.0045

0 0.5 1 1.5 2.0 2.5

Relativefr

equency

Probability (in millionths)

9080706050403020100

100

Cumulativefrequency%

Mean=

3,1

07

E-0

07

0.0080.5655% 90% 5%

Mean=

0.0

00160

00.00050.0010.00150.0020.00250.0030.00350.0040.0045

0.005

0 0.2 0.4 0.6 0.8 1 1.2

Relativefrequency

Number of infected embryos (in thousandths)

Fig. 5Possible number of years before foot and mouth disease virus is introduced into the USA via importation of cloned embryos

3.41 +5.0% 95.0% 0.0%

0

0.02

0.04

0.06

0.08

0.1

2.25 2.75 3.25 3.75 4.25 4.75 5.25 5.75 6.25

Relativefrequency

Number of years (Log. transformed)

Mean=

4.2

424

0.430.43

0.390.29

0.230.05

0.050.04

0.040.02

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

Coefficient value

C-O-COocyte washing

Embryo testing protocolsEmbryo washing

Oocyte donorCell culture treatment

Diagnostic test efficiencyCell testing protocolsEmbryo development

Cell donor

Fig. 3Probability density function (cumulative distribution) for the risk of introducing foot and mouth disease virus into the USA via clonedembryos created outside the USA

C-O-C: cumulusoocyte complex

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work demonstrating that conventionally produced (invitro-developed) bovine embryos, which may still containFMDV after a regular washing protocol (23), can berendered 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 transmittingFMDV is significantly affected by the status of C-O-Cs andtheir handling. This study may have erred towards an over-cautious approach to levels of risk in the somatic cell andC-O-C/oocyte donors. However, most abattoirs fromwhich C-O-Cs/oocytes are obtained perform thoroughante-mortem and post-mortem inspections, so viraemic(hence febrile) and clinically ill animals are unlikely to beselected as oocyte donors. In addition, as far as the authorsare aware, FMDV has never been found in the ovaries orC-O-Cs of chronically infected animals (17, 22).

Furthermore, sensitive tests can be used to detect FMDV inthe donors and in any degenerated embryos and washfluids, so that if infection is present the cells and oocytesare not used for cloned embryo production. It should alsobe noted that in persistently infected donor animals(carriers), the FMDV is usually located in the dorsal softpalate and in the pharyngeal roof above the soft palate(17). For SCNT procedures in cattle, differentiated musclecells (10) or fibroblasts obtained from skin or the earpunch (9, 41, 42) tend to be used, and so high-riskpalatine tissues can be avoided.

Taken together with the published results of experimentsinvolving in vivo and in vitro exposure of bovine embryosto FMDV, this work suggests that the risk of transmittingthe virus via the importation of cloned embryos isnegligible, even if the donors of the somatic cells and/orthe oocytes are persistently infected at the time ofcollection. The IETS has classified FMD as a Category-1disease, meaning that the risk of transmission via in vivo-derived bovine embryos is negligible, provided thatembryos are handled according to IETS protocols betweencollection and transfer (44). Thus, proper processing ofcells, oocytes and embryos (including exposure to trypsin),coupled with practices that ensure that donors are notacutely infected when cells are collected, should rendercloned embryos safe even if imported from FMD-affectedregions.

As far as the authors are aware, QRA has not been appliedpreviously to analyse the risk of introducing FMDV via themovement of infected cloned embryos. However, the

World Organisation for Animal Health (OIE) has also

concluded that international movement of cloned embryosis unlikely to lead to the release of adventitious infectiousagents (45). The OIE did note that the extensivemanipulation of donor cells, oocytes and embryos that is

involved in cloning by SCNT may exacerbate the risk oftransfer of adventitious agents and that the risks fromcloning are greater than those involved in in vitrofertilisation (IVF) procedures. In order to further minimiseany perceived risk associated with cloning by SCNT,practitioners should espouse the IETS recommendationthat the media in which the embryos are manipulatedbefore compromising the integrity of the ZP should besterile (37). If this can be attained, embryo production byin vitro techniques has low potential for transmittingdiseases (33, 36), and this can be applied in evaluatingrisks in SCNT (45).

Embryo production by SCNT (and IVF) also has one majorcomparative advantage. The production pathway providescontrol points and sufficient time to allow for each batch ofembryos produced to be monitored and assessed for theirhealth status. Such control begins with the strictmaintenance of medical records for the donor of the somaticcells. Furthermore, regular sampling of all the media used inthe process and any degenerated embryos would give a veryaccurate indication of the infectious milieu to which viableembryos may have been exposed (36).

In conclusion, according to this QRA, the risk of

introduction of FMDV into the USA via cloned bovineembryos is negligible and in the order of 3.1 10 7.However, to provide further experimental evidence tosubstantiate this conclusion, it would be necessary to usesomatic cells and oocytes from infected donors to producecloned animals and transfer them into recipients.

AcknowledgementsThis study was supported by funds provided by the UnitedStates Department of Agriculture, Animal and Plant HealthInspection Service through a cooperative agreement to

Tuskegee University, Tuskegee, Alabama.


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valuation quantitative du risque dintroduction du virus de lafivre aphteuse aux tats-Unis dAmrique lors dimportations

dembryons bovins clonsB. Asseged, B. Tameru, D. Nganwa, R. Fite & T. Habtemariam

RsumLa scurit des changes internationaux danimaux dlevage ou de leursproduits sappuie sur les informations obtenues concernant le risquedintroduction des agents infectieux, en particulier le virus de la fivre aphteuse.Bien que les voies de transmission de ce virus puissent varier, lexistence dunrisque dintroduction li aux importations dembryons clons a rcemment tsuspecte aux tats-Unis dAmrique. En consquence, un modle dvaluationquantitative du risque a t labor afin de dterminer, au moyen dun calcul des

probabilits, les scnarios pouvant donner lieu lintroduction puis au maintiendu virus de la fivre aphteuse suite limportation dembryons bovins clons. Laprobabilit dintroduction du virus de la fivre aphteuse par des embryonsclons, telle quelle a t estime au moyen du logiciel @RISK associ unesimulation de Monte-Carlo portant sur 50 000 itrations, sest leve 3,1 10 7.Compte tenu des protocoles en vigueur pour le clonage, et dans lhypothsedune importation annuelle de 250 1 700 embryons clons (moyenne = 520), lenombre escompt dembryons infects varie de 1,1 107 4,4 103 (moyenne= 1,6 104) par an. Lanalyse des voies critiques de transmission a montr que lerisque dintroduction du virus de la fivre aphteuse aux tats-Unis par cette voieest extrmement faible.

Mots-clsAnalyse des voies critiques de transmission Clonage tats-Unis dAmrique valuation quantitative du risque Fivre aphteuse Simulation de Monte-Carlo.

Evaluacin cuantitativa del riesgo de introduccindel virus de la fiebre aftosa en los Estados Unidosde Amrica a travs de embriones bovinos clonados

B. Asseged, B. Tameru, D. Nganwa, R. Fite & T. Habtemariam

ResumenEl comercio internacional de ganado bovino y sus derivados exige disponer dedatos sobre el riesgo de introduccin de agentes infecciosos como el virus de lafiebre aftosa (VFA). Aunque este virus puede transmitirse por diversas vas, enlos Estados Unidos de Amrica (EE.UU.) preocupa ltimamente la posibilidad deque penetre en el pas a travs de embriones clonados. Los autores describenun modelo de evaluacin cuantitativa del riesgo elaborado con el fin dedeterminar (con su correspondiente probabilidad matemtica) las eventualessituaciones que podran desembocar en la introduccin y permanencia del virus

a consecuencia de la importacin de embriones bovinos clonados. Utilizando elprograma informtico @RISK con una simulacin de Montecarlo que entraa50.000 iteraciones, se cifr en 3,1 107 la probabilidad de introduccin del VFA

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a travs de embriones clonados. Habida cuenta del protocolo actualmenteaplicado, y presuponiendo un volumen de importaciones anuales de entre 250 y1.700 (mediana = 520) embriones clonados, se calcula que el nmero anual deembriones infectados se sita entre 1,1 107 y 4,4 103 (mediana = 1,6 104).

El anlisis de rutas crticas dej patente un riesgo extremadamente bajo depenetracin del VFA en los EE.UU. por esta va.

Palabras claveAnlisis de rutas crticas Clonacin Estados Unidos de Amrica Evaluacincuantitativa del riesgo Fiebre aftosa Simulacin de Montecarlo.

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