Health and Safety Advisory Committee Research Update

278 Pages·2013·7.55 MB·English

HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS

****DISEASE
 TRANSMISSION
 BY
 IN
 VIVO
 PRODUCED
 EMBRYOS**** ................................6

Cattle.........................................................................................................................................................................................6

•
  Bovine
 immunodeficiency
 virus............................................................................................................................6

•
  Bovine
 leukemia
 virus
 (BLV)..................................................................................................................................6

•
  Bluetongue
 virus
 (BTV)............................................................................................................................................8

•
  Bluetongue
 virus
 serotype
 8................................................................................................................................12

•
  Infectious
 bovine
 rhinotracheitis/infectious
 pustular
 vaginitis
 virus
 (IBRV/IPVV/BHV-­‐1)..13

•
  Bovine
 Herpes
 virus
 -­‐1
 (BHV-­‐1).........................................................................................................................16

•
  Bovine
 herpesvirus
 4
 (BHV-­‐4)............................................................................................................................19

•
  Bovine
 herpesvirus
 5
 (BHV-­‐5)............................................................................................................................19

•
  Bovine
 viral
 diarrhea
 virus
 (BVDV)..................................................................................................................20

•
  Parvovirus
 (BPV)......................................................................................................................................................37

•
  Akabane
 virus
 (AV)..................................................................................................................................................37

•
  Foot
 and
 mouth
 disease
 virus
 (FMDV)............................................................................................................37

•
  Vesicular
 stomatitis
 virus
 (VSV)........................................................................................................................38

•
  Rinderpest
 virus
 (RPV)..........................................................................................................................................39

•
  Pseudorabies
 virus
 (PrV)......................................................................................................................................40

•
  Parainfluenza-­‐3
 virus
 (PI3V)...............................................................................................................................40

•
  Bovine
 enterovirus
 (BEV).....................................................................................................................................41

•
  Brucella
 abortus
 (B.
 abortus)..............................................................................................................................41

•
  Chlamydophila
 abortus
 (formerly:
 Chlamydia
 psittaci)..........................................................................45

•
  Histophilus
 somnusformerly
 Haemophilus
 somnus
 (H.
 Somnus)......................................................45

•
  Escherichia
 coli
 (E.coli)..........................................................................................................................................46

•
  Ureaplasma
 diversum
 (U.diversum)................................................................................................................46

•
  Leptospira
 spp...........................................................................................................................................................48

•
  Mycobacterium
 bovis..............................................................................................................................................48

•
  Mycoplasma
 spp........................................................................................................................................................49

•
  Mycobacterium
 avium
 subsp.
 paratuberculosis.........................................................................................50

•
  Neospora
 caninum...................................................................................................................................................52

•
  Bovine
 spongiform
 encephalopathy
 (BSE)
 agent.......................................................................................57

•
  Miscellaneous
 or
 multiple
 microorganisms..................................................................................................59

Sheep.....................................................................................................................................................................................60

•
  Border
 disease
 virus
 (BDV)
 and
 Bovine
 viral
 diarrhoea
 virus
 (BVDV)............................................60

•
  Bluetongue
 virus
 (BTV).........................................................................................................................................61

•
  Foot
 and
 mouth
 disease
 virus.............................................................................................................................63

•
  Scrapie
 agent..............................................................................................................................................................63

•
  Campylobacter
 fetus................................................................................................................................................67

•
  Chlamydophila
 abortus
 (formerly:
 Chlamydia
 psittaci)..........................................................................68

•
  Retroviruses................................................................................................................................................................68

•
  Maedi-­‐Visna
 virus
 (MVV)......................................................................................................................................69

•
  Sheep
 pulmonary
 adenomatosis
 (SPA)...........................................................................................................71

•
  Brucella
 ovis
 (B.ovis)..............................................................................................................................................72

•
  Brucella
 abortus
 (B.
 abortus)..............................................................................................................................73

Goats......................................................................................................................................................................................73

•
  Bluetongue
 virus.......................................................................................................................................................73

•
  Caprine
 arthritis-­‐encephalitis
 virus
 (CAEV).................................................................................................75

•
  Foot
 and
 mouth
 disease
 virus.............................................................................................................................80

•
  Coxiella
 burnetii........................................................................................................................................................80

•
  Mycoplasma
 mycoides
 mycoides
 (LC
 type)..................................................................................................81

2012 IETS research update 1
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
•
  Bovine
 spongiform
 encephalopathy
 agent
 (BSE).......................................................................................81

•
  Scrapie...........................................................................................................................................................................82

Pigs.........................................................................................................................................................................................82

•
  Porcine
 circovirus.....................................................................................................................................................82

•
  Parvovirus
 (PPV)......................................................................................................................................................83

•
  Porcine
 reproductive
 and
 respiratory
 syndrome
 virus
 (PRRSV)........................................................85

•
  Pseudorabies
 virus
 (PrV)/Aujeszky's
 Disease.............................................................................................90

•
  African
 swine
 fever
 virus
 (ASFV).......................................................................................................................95

•
  Enteroviruses
 (ECPO-­‐3;
 ECPO-­‐6)......................................................................................................................95

•
  Foot
 and
 mouth
 disease
 virus
 (FMDV)............................................................................................................95

•
  Swine
 vesicular
 disease
 (SVD)............................................................................................................................96

•
  Hog
 cholera
 virus
 (HCV);
 classical
 swine
 fever
 virus................................................................................96

•
  Vesicular
 stomatitis
 virus
 (VSV)........................................................................................................................97

•
  Porcine
 Rubulavirus
 (PoRV;
 causative
 agent
 of
 blue
 eye
 disease).....................................................98

•
  Mycoplasma
 hyopneumonia,
 Streptococcus
 suis,
 Pasteurella
 multocida........................................98

•
  Chlamydophila/Chlamydia...................................................................................................................................98

Horses....................................................................................................................................................................................99

•
  Equine
 Herpes
 Virus
 1
 (EHV1)...........................................................................................................................99

•
  Equid
 herpesvirus
 3...............................................................................................................................................101

•
  Taylorella
 equigenitalis
 (causative
 agent
 of
 contagious
 equine
 metritis
 [CEM])........................101

•
  Equine
 arteritis
 virus............................................................................................................................................102

Human.................................................................................................................................................................................102

Hamster..............................................................................................................................................................................103

Mice......................................................................................................................................................................................103

•
  Lymphocytic
 choriomeningitis
 virus
 (LCMV)............................................................................................104

•
  Mouse
 Parvovirus
 (MPV)....................................................................................................................................105

****DISEASE
 TRANSMISSION
 THROUGH
 IN
 VITRO
 PRODUCED
 EMBRYOS****...............106

Cattle....................................................................................................................................................................................106

•
  Akabane
 virus...........................................................................................................................................................106

•
  Bluetongue
 virus.....................................................................................................................................................106

•
  Bovine
 herpes
 virus
 1
 (BoHV-­‐1)......................................................................................................................107

•
  Bovine
 herpesvirus-­‐4............................................................................................................................................107

•
  Bovine
 herpesvirus-­‐5............................................................................................................................................107

•
  Bovine
 immunodeficiency
 virus
 (BIV)..........................................................................................................109

•
  Bovine
 leukemia
 virus..........................................................................................................................................110

•
  Infectious
 bovine
 rhinotracheitis
 virus/bovine
 herpesvirus
 -­‐1.........................................................111

•
  Bovine
 virus
 diarrhoea
 virus
 (BVDV)............................................................................................................119

•
  BVDV
 and
 BHV-­‐1.....................................................................................................................................................147

•
  Bluetongue
 virus
 serotype
 8..............................................................................................................................148

•
  Enzootic
 hemorrhagic
 disease..........................................................................................................................149

•
  Foot-­‐and-­‐mouth
 disease
 virus..........................................................................................................................150

•
  Campylobacter
 fetus
 (C.
 fetus)..........................................................................................................................151

•
  Leptospira
 spp.........................................................................................................................................................151

•
  Mycoplasma
 spp......................................................................................................................................................152

•
  Neospora
 caninum.................................................................................................................................................153

•
  Trichomonas
 fetus..................................................................................................................................................155

•
  Mycobacterium
 avium
 ssp.
 paratuberculosis.............................................................................................156

•
  Miscellaneous
 infections
 (and
 mixed
 agents)
 or
 multiple
 microorganisms.................................157

•
  Genetic
 diseases......................................................................................................................................................160

•
  Quality
 control
 testing..........................................................................................................................................160

2012 IETS research update 2
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
Goats....................................................................................................................................................................................161

•
  Caprine
 arthritis-­‐encephalitis
 virus
 (CAEV)...............................................................................................161

Horses..................................................................................................................................................................................164

•
  Equine
 arteritis
 virus............................................................................................................................................164

•
  Equine
 infectious
 anemia
 virus........................................................................................................................166

Pigs.......................................................................................................................................................................................167

•
  Hog
 cholera
 virus
 (HCV)
 (classic
 swine
 fever
 virus)...............................................................................167

•
  Multiple
 viruses
 (including
 EMCV,
 PCV2,
 PPV
 PRRSV
 and
 BVDV)....................................................167

•
  Porcine
 circovirus...................................................................................................................................................168

•
  Porcine
 reproductive
 and
 respiratory
 syndrome
 virus
 (PRRSV)......................................................169

•
  Pseudorabies
 virus.................................................................................................................................................170

•
  Chlamydophila/Chlamydia.................................................................................................................................171

Sheep...................................................................................................................................................................................172

•
  Maedi-­‐Visna
 Virus
 (MVV)....................................................................................................................................172

Rat.........................................................................................................................................................................................173

•
  Antiviral
 treatments
 (rat
 embryos)................................................................................................................173

Human.................................................................................................................................................................................174

•
  Human
 (Hepatitis
 B&C,
 HIV,
 Rubella)............................................................................................................174

Rabbits................................................................................................................................................................................175

Mice......................................................................................................................................................................................176

•
  Mouse
 Minute
 Virus
 (MMV)...............................................................................................................................176

•
  Mouse
 minute
 virus
 and
 &
 Mouse
 Hepatitis
 Virus...................................................................................177

•
  HIV-­‐1
 (AIDS).............................................................................................................................................................178

•
  P.
 pneumotropica
 and
 P.
 ureae.........................................................................................................................178

•
  Neospora
 caninum.................................................................................................................................................178

•
  Ecotropic
 murine
 leukemia
 viruses
 (MuLV)...............................................................................................179

•
  MHV..............................................................................................................................................................................179

Bison....................................................................................................................................................................................180

EMBRYO
 DISINFECTION.............................................................................................................................................180

•
  Biosecurity.................................................................................................................................................................180

•
  Trypsin
 treatment..................................................................................................................................................181

•
  Other
 treatments.....................................................................................................................................................185

***Miscellaneous***.....................................................................................................187

•
  Antimicrobials/Antibiotics.................................................................................................................................187

•
  Antiviral
 treatments..............................................................................................................................................187

•
  Contaminating
 microorganisms.......................................................................................................................194

•
  Cryopreservation
 &
 Disinfection
 of
 Dry
 Shippers....................................................................................196

•
  Culture
 systems
 free
 of
 animal-­‐origin
 supplements...............................................................................199

•
  Diagnostic
 assays
 on
 embryos..........................................................................................................................199

•
  Diagnosis
 using
 Multiplex
 testing....................................................................................................................200

•
  Disinfection
 of
 fluids..............................................................................................................................................201

•
  Endogenous
 retroviral
 sequences...................................................................................................................202

•
  Exogenous
 DNA
 uptake........................................................................................................................................202

•
  Fetal
 bovine
 serum.................................................................................................................................................203

•
  Genetic
 defects.........................................................................................................................................................204

•
  Immunoglobulins
 in
 body
 fluids......................................................................................................................206

•
  Interspecies
 embryos............................................................................................................................................206

•
  Miscellaneous
 Topics
 (
 media
 altern.,
 ethylene
 oxide
 toxicity
 ,
 &
 ZP
 invasion)..........................207

•
  Pathogens
 in
 Materials
 of
 Animal
 Origin......................................................................................................208

•
  Risk
 Assessment......................................................................................................................................................210

•
  Sanitary/Quality
 controls...................................................................................................................................212

2012 IETS research update 3
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
•
  Viral
 infection
 effects
 on
 oocytes.....................................................................................................................214

•
  Vitrification...............................................................................................................................................................215

•
  Zona
 pellucida..........................................................................................................................................................216

***Semen
 (since
 2010)***.............................................................................................220

Cattle....................................................................................................................................................................................220

•
  Bluetongue
 virus.....................................................................................................................................................220

•
  Bovine
 herpesvirus................................................................................................................................................221

•
  Bovine
 viral
 diarrhea
 virus
 (BVDV)................................................................................................................221

•
  Semen
 treatments..................................................................................................................................................223

Goats....................................................................................................................................................................................225

•
  Bluetongue
 virus.....................................................................................................................................................225

Horses..................................................................................................................................................................................225

•
  Equine
 herpesvirus
 types
 1
 (EHV-­‐1)
 and
 4
 (EHV-­‐4)...............................................................................225

•
  Various
 pathogens..................................................................................................................................................226

Humans
 and
 bovine.......................................................................................................................................................228

HIV
 and
 HCV ..................................................................................................................228

REFERENCES...................................................................................................................229

•
  A.....................................................................................................................................................................................229

•
  B......................................................................................................................................................................................230

•
  C......................................................................................................................................................................................237

•
  D.....................................................................................................................................................................................238

•
  E......................................................................................................................................................................................239

•
  F......................................................................................................................................................................................240

•
  G......................................................................................................................................................................................241

•
  H.....................................................................................................................................................................................246

•
  I.......................................................................................................................................................................................247

•
  J.......................................................................................................................................................................................248

•
  K.....................................................................................................................................................................................248

•
  L......................................................................................................................................................................................249

•
  M.....................................................................................................................................................................................250

•
  N.....................................................................................................................................................................................254

•
  O.....................................................................................................................................................................................254

•
  P......................................................................................................................................................................................255

•
  Q.....................................................................................................................................................................................256

•
  R.....................................................................................................................................................................................256

•
  S......................................................................................................................................................................................257

•
  T......................................................................................................................................................................................262

•
  U.....................................................................................................................................................................................263

•
  V......................................................................................................................................................................................263

•
  W....................................................................................................................................................................................265

•
  X......................................................................................................................................................................................267

•
  Y......................................................................................................................................................................................267

•
  Z......................................................................................................................................................................................267

REVIEW
 PAPERS
 ON
 EMBRYO
 TRANSFER
 -­‐
 INFECTIOUS
 DISEASE
 TRANSMISSION.....................267

•
  A.....................................................................................................................................................................................267

•
  B......................................................................................................................................................................................268

•
  C......................................................................................................................................................................................269

•
  D.....................................................................................................................................................................................269

•
  E......................................................................................................................................................................................269

2012 IETS research update 4
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
•
  F......................................................................................................................................................................................270

•
  G......................................................................................................................................................................................270

•
  H.....................................................................................................................................................................................270

•
  IJK..................................................................................................................................................................................271

•
  L......................................................................................................................................................................................271

•
  M.....................................................................................................................................................................................271

•
  N.....................................................................................................................................................................................272

•
  O.....................................................................................................................................................................................272

•
  P......................................................................................................................................................................................273

•
  Q.....................................................................................................................................................................................273

•
  R.....................................................................................................................................................................................273

•
  S......................................................................................................................................................................................274

•
  T......................................................................................................................................................................................276

•
  U.....................................................................................................................................................................................277

•
  V......................................................................................................................................................................................277

•
  W....................................................................................................................................................................................277

2012 IETS research update 5
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
****DISEASE
 TRANSMISSION
 BY
 IN
 VIVO
 PRODUCED
 EMBRYOS****

Cattle

• Bovine
 immunodeficiency
 virus

Three
  experiments
  were
  conducted
  to
  determine
  whether
  the
  lentivirus,
  bovine

immunodeficiency
 virus
 (BIV)
 is
 likely
 to
 be
 transmitted
 via
 embryo
 transfer.
 In
 the
 first

experiment,
 embryos
 collected
 from
 BIV-­‐negative
 heifers
 were
 exposed
 in
 vitro
 to
 BIV

for
  24
  h,
  washed
  and
  then
  tested
  for
  the
  presence
  of
  the
  provirus.
  In
  the
  second

experiment,
  embryos
  obtained
  from
  BIV-­‐negative
  heifers
  were
  transferred
  to
  the

uterine
 horns
 of
 BIV-­‐infected
 heifers;
 24
 h
 later
 these
 embryos
 were
 recovered
 and

tested
 for
 the
 presence
 of
 BIV.
 In
 the
 third
 experiment,
 embryos
 were
 collected
 from

heifers
  experimentally
  infected
  with
  BIV
  and
  then
  transferred
  to
  BIV-­‐negative

recipients.
 In
 all
 three
 experiments,
 (BIV)
 proviral
 DNA
 was
 not
 detected
 by
 PCR
 in

association
  with
  any
  oocytes,
  embryos,
  follicular
  fluid,
  oviductal
  or
  uterine
  washes.

Twelve
  single
  embryos
  collected
  from
  BIV
  experimentally
  infected
  donors
  were

transferred
 to
 BIV-­‐negative
 recipients
 resulting
 in
 the
 birth
 of
 7
 calves
 all
 of
 which
 were

also
 negative
 for
 BIV;
 the
 recipients
 remained
 BIV-­‐negative
 throughout
 the
 experiment.

In
 conclusion,
 this
 study
 demonstrates
 that
 it
 is
 possible
 to
 produce
 transferrable
 stage

embryos
 from
 donors
 infected
 with
 BIV
 and
 that
 such
 embryos
 are
 unlikely
 to
 transmit

this
  agent
  either
  to
  the
  recipients
  or
  the
  resulting
  offspring.
  (Bielanski
  A,
  et
  al.

Theriogenology
 2001;55:641-­648.)

• Bovine
 leukemia
 virus
 (BLV)

A
 summarization
 of
 embryo
 transfer
 results
 (Di
 Giacomo
 et
 al,
 1986,
 1990;
 Eaglesome
 et

al,
 1982;
 Hare
 et
 al,
 1985;
 Kaja
 et
 al,
 1984;
 Krolinski
 et
 al,
 1992;
 Olson
 et
 al,
 1982;
 Parodi

et
 al
 1983;
 Steffani
 et
 al,
 1987;
 Coulthard,
 unpublished
 data)
 shows
 that
 over
 1300
 zona

pellucida-­‐intact
 (ZP-­‐I)
 embryos
 have
 been
 collected
 from
 BLV-­‐seropositive
 dams
 bred

by,
 or
 inseminated
 with
 semen
 from,
 either
 BLV-­‐seropositive
 or
 BLV-­‐seronegative
 sires,

washed
  and
  then
  transferred
  to
  BLV-­‐seronegative
  recipients.
  The
  transfers
  have

produced
  over
  618
  pregnancies
  with
  599
  live
  calves.
  None
  of
  the
  recipients

seroconverted
 and
 all
 of
 the
 calves
 remained
 seronegative.

BLV
 was
 not
 identified
 from
 60
 ZP-­‐I
 embryos
 and
 26
 ZP-­‐I
 unfertilized
 eggs,
 collected

from
 BLV-­‐seropositive
 donors,
 washed
 and
 then
 co-­‐cultivated
 with
 fetal
 lamb
 spleen

cells,
 based
 on
 syncytium
 induction
 and
 immunofluorescence.
 (Bouillant
 et
 al,
 Ann

Rech
 Vet
 1981;12:385-­395.)

BLV
 was
 identified
 using
 the
 above
 procedures
 in
 4/25
 (16%)
 flush
 fluids
 from
 BLV-­‐
seropositive
 donors,
 probably
 as
 a
 result
 of
 blood
 cell
 contamination.
 (Bouillant
 et
 al,

Ann
 Rech
 Vet
 1981;12:385-­395.)

Twenty-­‐four,
  day
  6,
  untreated
  ZP-­‐I
  embryos;
  10,
  day
  6,
  pronase
  treated,
  ZP-­‐I
  (but

weakened)
 embryos,
 and
 14,
 day
 12,
 hatched
 embryos,
 collected
 from
 BLV-­‐seronegative

2012 IETS research update 6
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
donors
 that
 were
 inseminated
 with
 semen
 from
 BLV-­‐seronegative
 bulls,
 were
 washed

and
 then
 layered
 onto
 cultures
 of
 BLV-­‐infected
 fetal
 lamb
 kidney
 cells
 for
 24
 hrs
 at
 37°C.

They
 were
 washed
 again
 and
 then
 transferred
 in
 the
 above
 groups
 to
 three
 recipients.

No
 pregnancies
 resulted
 and
 the
 recipients
 did
 not
 seroconvert,
 nor
 could
 BLV-­‐antigen

be
 detected
 up
 to
 120
 days
 post
 transfer.
 (Hare
 et
 al,
 Can
 Vet
 J
 1985;26:231-­234.)

Day
 7
 embryos
 were
 collected
 non-­‐surgically
 and
 washed
 3X
 prior
 to
 being
 bisected
 and

transferred
 to
 recipient
 animals
 (not
 stated
 if
 bisected
 embryos
 were
 transferred
 naked

or
 within
 a
 ZP).
 Sixty
 split
 embryo
 calves
 resulted.
 Of
 these,
 19
 originated
 from
 BLV
+ve

dams
 and
 BLV sires,
 4
 from
 BLV sires
 and
 BLV
  dams,
 9
 from
 BLV dams
 and

-­‐ve
  +ve
  -­‐ve
  +ve

sires,
 and
 28
 from
 BLV dairy
 herd.
 All
 calves
 were
 repeatedly
 tested
 serologically

-­‐ve

negative
  for
  BLV
  and
  all
  recipients
  remained
  serologically
  negative.
  (Lorton
  et
  al,

Theriogenology
 1987;27:250.)

Thirteen
 hundred
 and
 six,
 fresh
 and
 frozen-­‐thawed,
 embryos,
 in
 approximately
 20%
 of

which
 the
 ZP
 was
 damaged,
 were
 transferred
 to
 recipients
 that
 were
 seronegative
 for

BLV.
 The
 embryos
 were
 collected
 from
 dairy
 herds
 where
 a
 majority
 of
 the
 cattle
 were

probably
 seropositive
 for
 BLV,
 although
 the
 exact
 level
 of
 infection
 was
 not
 known

because
 prior
 health
 testing
 for
 this
 disease
 was
 not
 required.
 (No
 washing
 procedure

for
 the
 embryos
 was
 described).
 All
 of
 the
 recipients
 remained
 seronegative
 for
 BLV.

(Thibier
 and
 Nibart,
 Theriogenology
 1987;27:37-­47.)

In
 a
 field
 study
 report,
 21
 seronegative
 calves
 were
 born
 as
 a
 result
 of
 embryos
 collected

from
 eight
 seropositive
 donors
 and
 transferred
 to
 20
 seronegative
 recipients.
 One
 of

four
 calves
 born
 as
 a
 result
 of
 embryos
 collected
 from
 a
 donor
 that
 was
 seronegative
 one

month
 before
 collection,
 but
 seropositive
 at
 the
 next
 test
 approximately
 nine
 months

later,
 and
 transferred
 to
 a
 recipient
 that
 was
 seronegative
 15-­‐20
 days
 before
 transfer,

but
 seropositive
 after
 parturition,
 was
 seropositive
 at
 birth.
 No
 mention
 is
 made
 in
 the

report
 of
 washing
 the
 embryos.
 The
 donors
 came
 from
 farms
 where
 the
 level
 of
 BLV

infected
 cattle
 ranged
 from
 35-­‐45%.
 There
 is
 no
 record
 of
 the
 level
 of
 BLV
 infection
 in

the
 herds
 of
 origin
 of
 the
 recipients.
 This
 report
 provides
 a
 good
 illustration
 of
 the

importance
 of:

a)
 the
 proper
 washing
 of
 embryos
 between
 collection
 and
 transfer

b)
 the
 quarantining
 and
 retesting
 of
 seronegative
 donors
 coming
 from
 infected
 premises

for
 an
 adequate
 time
 period
 prior
 to
 collection

c)
 the
 quarantining
 and
 retesting
 of
 seronegative
 recipients
 for
 an
 adequate
 time
 period

prior
 to
 transfer
 and
 their
 isolation
 from
 positive
 animals
 throughout
 gestation.

(Severini
 et
 al,
 Atti
 Soc
 Italiana
 Buiatria
 1984;15:481-­488.)

Embryos
 were
 transferred
 from
 28
 Black
 Pied
 cows
 that
 were
 serologically
 positive
 for

BLV
 to
 44
 seronegative
 recipients.
 The
 efficacy
 of
 controlling
 leukosis
 by
 means
 of

embryo
  transfer
  was
  confirmed.
  (Korolev
  et
  al,
  Trudy
  Vsesoyuznogo
  Instituta

Eksperimental
 ‘Noi
 Veterinarii
 1987;64:59-­62.)

2012 IETS research update 7
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
The
 stated
 purpose
 of
 the
 study
 was
 to
 "confirm
 the
 possibility
 of
 protection
 of
 calves
 of

high
  breeding
  value
  of
  leukaemic
  cows
  against
  infections
  with
  EBLV
  using
  embryo

transfer
 methods.

"From
 152
 donors
 1390
 embryos
 were
 obtained
 and
 only
 676
 (48.6%)
 were
 qualified
 as

suitable
  for
  transfer.
  The
  amount
  and
  types
  of
  cellular
  elements
  in
  10
  consecutive

dilutions
 of
 16
 tests
 of
 flushing
 from
 the
 uterus
 of
 infected
 cows
 were
 assessed.
 In
 1
 ml

of
 uteral
 flushing,
 an
 average
 of
 1.6
 million
 various
 morfotic
 elements
 were
 found.
 In
 the

first
 and
 sporadically
 in
 the
 second
 dilutions
 these
 cells
 were
 observed.
 From
 the
 next

solutions
  no
  cellular
  formations
  were
  found.
  During
  histological
  investigations,
  the

lymphoid
 cells
 were
 observed
 neither
 on
 the
 zona
 pellucida
 of
 embryos
 nor
 in
 their

vicinity.
 Then
 585
 embryos
 from
 the
 leukaemic
 donors
 were
 rinsed
 four
 times
 and

transferred
 resulting
 in
 278
 pregnant
 cows.
 There
 274
 calves
 were
 born
 free
 from

leukaemia
 with
 four
 dying
 during
 parturition."
 (gel
 precipitation
 test
 or
 ELISA
 were

tests
 used
 on
 calves)
 (Krolinski
 J,
 et
 al.,
 Medycyna
 Weterynaryjna
 1992;48:79-­81

and
  Krolinski
  J,
  et
  al.,
  Proceed
  10e
  Reunion
  A.E.T.A.
  -­
  Lyon,
  9-­10
  September,

1994:page198.)

A
 case
 was
 discovered
 where
 the
 embryo
 transfer
 (ET)
 calf
 had
 been
 infected
 with

bovine
 leukemia
 virus
 (BLV)
 from
 the
 recipient
 cow.
 The
 embryo
 was
 transferred
 from

the
 BLV-­‐uninfected
 donor
 cow
 to
 the
 recipient
 cow.
 However,
 the
 BLV
 test
 had
 not
 been

performed
 on
 the
 recipient
 cow
 before
 ET
 was
 performed.
 The
 ET
 calf
 was
 raised
 in
 a

calf
 hutch
 from
 birth
 to
 1-­‐month
 old
 and
 was
 given
 the
 recipient
 cow's
 colostrum
 and

milk
 artificially.
 The
 ET
 calf
 was
 raised
 with
 the
 two
 other
 calves
 from
 a
 1-­‐month
 old
 to
 a

6-­‐month
 old.
 The
 BLV
 test
 was
 performed
 on
 the
 ET
 calf
 by
 agar
 gel
 precipitation
 (AGP)

and
 passive
 haemagglutination
 (PHA)
 assay
 when
 the
 ET
 calf
 was
 6
 months
 old.
 Because

the
 ET
 calf
 was
 positive,
 the
 BLV
 test
 was
 performed
 on
 the
 recipient
 cow,
 the
 two
 other

calves
 raised
 with
 the
 ET
 calf
 and
 the
 two
 dams
 of
 the
 two
 other
 calves.
 Because
 the

recipient
 cow
 only
 was
 positive
 at
 the
 time
 of
 the
 first
 test,
 we
 judged
 that
 the
 ET
 calf

had
 been
 infected
 with
 BLV
 from
 the
 recipient
 cow.
 The
 importance
 of
 the
 BLV
 test

being
 carried
 out
 on
 the
 recipient
 cow
 for
 the
 prevention
 of
 enzootic
 bovine
 leukemia
 in

a
 case
 of
 ET
 was
 recognised.
 (Fukai
 K,
 et
 al.,
 Zentralbl
 Veterinarmed
 (J
 Vet
 Med)

1999;46:511-­515.)

• Bluetongue
 virus
 (BTV)

BTV
 did
 not
 penetrate
 the
 zona
 pellucida
 (ZP)
 or
 attach
 to
 it
 when
 day
 5
 to
 7,
 ZP-­‐I

embryos
 (n
 =
 120)
 were
 exposed
 to
 10 -­‐10 pfu/ml
 of
 virus
 (serotype
 10)
 for
 1-­‐24
 hrs,

2 7

washed
  and
  then
  assayed.
  Nor
  was
  the
  embryonic
  development
  of
  these
  embryos

affected
 by
 exposure
 to
 the
 virus.
 (Singh
 et
 al,
 Theriogenology
 1982;17:437-­444.)

Thirty-­‐six
 6-­‐7
 day
 old
 bovine
 embryos
 from
 BTV
 seronegative
 cows
 were
 exposed
 to

0.75ml
  of
  a
  BTV
  (serotype
  11)
  suspension
  containing
  TCID 10 /0.1ml
  for
  18-­‐24

50
  5.6
hours.
 Five
 embryos
 were
 prepared
 for
 electron
 microscopy
 examination
 (EM)
 without

any
 washing:
 three
 of
 the
 five
 embryos
 had
 numerous
 particles
 on
 the
 surface
 of
 the

zona
  pellucida
  but
  no
  evidence
  of
  particles
  within
  the
  embryo.
  After
  5
  washes
  in

0.75ml/wash,
 nine
 of
 21
 exposed
 embryos
 were
 positive
 in
 bovine
 turbinate
 cell
 culture

based
 upon
 production
 of
 a
 cytopathic
 effect.
 Eight
 of
 ten
 embryos
 taken
 from
 the
 5th

2012 IETS research update 8
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
wash
 after
 12
 hrs
 and
 examined
 by
 EM
 had
 viral
 particles
 on
 the
 surface
 of
 the
 zona

pellucida,
 but
 no
 particles
 were
 observed
 within
 the
 embryo.
 (Gillespie
 et
 al,
 Dtsch

tierarztl
 Wschr
 1990;97:65-­68.)

Twenty-­‐one
 6-­‐7
 day
 old
 bovine
 embryos
 from
 BTV
 seronegative
 cows
 were
 placed
 in
 a

bovine
  turbinate
  cell
  culture
  containing
  0.75ml
  of
  media
  which
  was
  seeded
  a
  few

minutes
 later
 with
 serotype
 11
 BTV
 at
 a
 calculated
 ratio
 of
 one
 infectious
 virus
 particle

for
 each
 cell
 (multiplicity
 of
 1:1).
 After
 18-­‐24
 hours
 in
 cell
 culture,
 nine
 unwashed

embryos
 were
 processed
 for
 and
 examined
 by
 EM:
 all
 exposed
 embryos
 had
 numerous

particles
  on
  the
  surface
  of
  the
  zona
  pellucida,
  but
  none
  were
  observed
  within
  the

embryo.
 After
 5
 washes
 containing
 0.75ml
 of
 media
 11
 of
 12
 exposed
 embryos
 caused
 a

cytopathic
 effect
 in
 cell
 culture.
 After
 exposure
 for
 12
 hours
 in
 the
 5th
 wash
 cell
 culture,

11
 of
 12
 embryos
 also
 had
 numerous
 particles
 on
 the
 surface
 of
 zona
 pellucida,
 but
 none

were
 observed
 within
 the
 embryo.
 (Gillespie
 et
 al,
 Dtsch
 tierarztl
 Wschr
 1990;97:65-­
68.)

Embryos
 were
 microinjected
 with
 "50
 picolitres"
 (TCID 10 )
 of
 virus,
 washed
 10x

50
  0.33
(0.75ml/wash),
 placed
 in
 bovine
 turbinate
 cell
 culture
 for
 18-­‐24
 hrs,
 exposed
 to
 BTV

antiserum
 for
 1
 hr
 and
 then
 washed
 5x.
 Cells
 were
 then
 removed
 from
 each
 embryo
 that

had
 an
 intact
 ZP.
 Cells
 were
 placed
 in
 BT
 cell
 culture
 for
 7
 days,
 followed
 by
 three
 blind

passages
 of
 7
 days
 if
 the
 primary
 culture
 showed
 no
 CPE.
 Most
 embryos
 were
 also

examined
 by
 EM.
 None
 of
 eight
 embryos
 were
 positive
 on
 cell
 culture
 and
 the
 one

embryo
 examined
 by
 EM
 was
 also
 negative.
 However
 results
 are
 inconclusive,
 because

less
 than
 one
 TCID of
 virus
 was
 present
 in
 "
 50
 picoliters
 of
 inoculum.
 (Gillespie
 et
 al,

50

Dtsch
 tierarztl
 Wschr
 1990;97:65-­68.)

Exposure
 to
 BTV
 (serotypes
 11,
 17)
 of
 day
 6-­‐1/2
 to
 7
 embryos
 (n
 -­‐
 18)
 from
 which
 ZP

had
 been
 removed
 by
 immersion
 in
 solution
 at
 pH
 2
 resulted
 in
 the
 accumulation
 of
 BTV

antigen
 in
 the
 blastomeres
 and
 embryonic
 death.
 The
 development
 of
 control
 embryos

(n
 =
 3)
 did
 not
 appear
 to
 be
 affected
 by
 immersion
 in
 solution
 at
 pH
 2.
 (Bowen
 et
 al,
 Am

J
 Vet
 Res
 1982;43:1907-­1911.)

Forty-­‐eight,
 day
 6-­‐8,
 ZP-­‐I
 embryos,
 collected
 from
 17
 BT-­‐viremic
 (serotypes
 10,
 11,
 13,

17,
  18)
  dams
  inseminated
  with
  BTV-­‐negative
  semen,
  were
  transferred
  to
  48
  BTV-­‐
seronegative
 recipients.
 Twenty-­‐five
 of
 the
 48
 recipients
 were
 pregnant
 at
 60
 days
 at

which
 time
 11
 pregnancies
 were
 terminated.
 There
 were
 4
 stillbirths
 and
 10
 live
 births.

All
 calves
 and
 recipients
 remained
 seronegative
 for
 BTV.
 Calves
 and
 recipients
 sampled

for
 BTV
 isolation
 were
 also
 negative.
 (Bowen
 et
 al,
 Am
 J
 Vet
 Res
 1983;44:1625-­1628

and
 Thomas
 et
 al,
 Theriogenology
 1983;19:425-­431.)

Twenty,
 day
 6,
 ZP-­‐I
 embryos,
 collected
 from
 three
 BTV-­‐seronegative
 dams,
 inseminated

with
  BTV-­‐positive
  semen
  (serotype
  17),
  were
  transferred
  to
  16
  BTV-­‐seronegative

recipients.
 Two
 of
 the
 donor
 heifers
 became
 infected.
 Eighteen
 of
 the
 20
 embryos
 were

from
 the
 two
 viremic
 donors
 and
 resulted
 in
 nine
 of
 the
 10
 pregnancies
 obtained.
 One

recipient
 aborted
 at
 3
 months.
 Nine
 recipients
 carried
 their
 calves
 to
 term.
 Six
 of
 the

nine
 delivered
 live
 healthy
 calves,
 one
 was
 euthanized
 during
 dystocia.
 One
 set
 of
 twins

and
 one
 single
 calf
 died
 during
 calving.
 None
 of
 the
 16
 recipients
 nor
 any
 of
 the
 calves

developed
 antibodies
 to
 BTV.
 Nor
 was
 BTV
 isolated
 from
 any
 of
 the
 uterine
 flush
 fluids.

(Thomas
 et
 al,
 Theriogenology
 1985;24:345-­350.)

2012 IETS research update 9
HASAC RESEARCH UPDATE - DISEASE TRANSMISSION BY EMBRYOS
Two
 superovulated
 seronegative
 cows
 were
 each
 inseminated
 three
 times
 at
 12-­‐hour

intervals
 with
 virus-­‐laden
 semen
 containing
 TCID 10 /0.1ml
 of
 BTV.
 The
 cows
 were

50
  5.8
flushed
 for
 embryo
 production
 6
 days
 after
 the
 last
 insemination.
 One
 cow
 produced
 7

embryos
 and
 the
 other
 one
 failed
 to
 provide
 any
 embryos.
 Attempts
 to
 demonstrate

virus
 in
 or
 on
 the
 embryos
 by
 EM
 or
 isolation
 by
 3
 blind
 transfers
 in
 cell
 culture
 failed.

After
 flushing
 the
 two
 cows
 were
 euthanized
 and
 many
 tissues,
 particularly
 from
 the

reproductive
 tract,
 were
 harvested
 and
 tested
 for
 presence
 of
 virus
 in
 cell
 culture.
 The

results
 were
 highlighted
 by
 the
 isolation
 of
 virus
 in
 cell
 culture
 from
 the
 ovary,
 uterine

horn,
  cervix
  and
  blood
  of
  each
  animal.
  (Schlafer
  et
  al,
  Dtsch
  tierarztl
  Wschr

1990;97:68-­72.)

Three
 superovulated
 seronegative
 cows
 were
 inseminated
 3
 times
 at
 12-­‐hour
 intervals

with
 semen
 from
 a
 seronegative
 bull.
 Immediately
 after
 the
 last
 insemination
 each
 cow

was
 inoculated
 intramuscularly
 with
 1ml
 of
 stock
 BTV
 containing
 TCID 10 /0.1ml.

50
  5.8
Five
 days
 later
 each
 cow
 was
 flushed
 for
 embryos.
 One
 cow
 had
 2
 embryos
 and
 no
 virus

could
 be
 demonstrated
 in
 these
 embryos
 by
 EM
 or
 in
 cell
 culture
 (3
 blind
 transfers).
 The

other
  2
  failed
  to
  produce
  embryos.
  Immediately
  after
  flushing,
  the
  3
  cows
  were

euthanized
 and
 tissues
 harvested
 for
 virus
 isolation.
 No
 virus
 was
 isolated
 from
 the

reproductive
 tract
 or
 blood
 of
 the
 cow
 that
 produced
 2
 embryos.
 Virus
 was
 isolated
 in

cell
 culture
 from
 the
 blood
 and
 at
 least
 one
 reproductive
 tissue
 of
 the
 other
 2
 cows.

(Schlafer
 et
 al,
 Dtsch
 tierarztl
 Wschr
 1990;97:68-­72.)

Three
  synchronized
  seronegative
  heifers
  were
  each
  implanted
  with
  2
  normally-­‐
developing
 6-­‐day-­‐old
 bovine
 embryos
 that
 had
 been
 kept
 in
 an
 infected
 cell
 culture
 for

24
 hours
 and
 subsequently
 washed
 3
 times
 (10ml
 each
 wash).
 None
 of
 the
 heifers

conceived
 but
 virus
 was
 isolated
 in
 cell
 culture
 from
 the
 vaginal
 swab
 and
 blood
 sample

taken
 on
 day
 7
 after
 implantation
 from
 1
 heifer
 only.
 The
 progesterone
 levels
 at
 4
 and
 7

weeks
 were
 normal.
 Two
 heifers
 were
 euthanized
 at
 54
 days
 after
 embryo
 transfer
 and

virus
 was
 not
 demonstrated
 in
 the
 blood
 and
 other
 tissues
 harvested
 at
 this
 time.
 All
 3

heifers
 seroconverted
 with
 serum
 neutralizing
 antibody
 titres
 ranging
 from
 8
 to
 32
 at

day
 35
 when
 tested
 against
 100
 TCID of
 BTV
 in
 the
 bovine
 turbinate
 cell
 culture

50

system.
 (Schlafer
 et
 al,
 Dtsch
 tierarztl
 Wschr
 1990;97:68-­72.)

Nineteen,
 day
 10-­‐11,
 hatched
 embryos,
 collected
 from
 10
 BT-­‐viremic
 (serotypes
 10,
 11,

13,
 17)
 dams
 inseminated
 with
 semen
 from
 BTV-­‐seronegative
 bulls,
 were
 transferred
 to

19
 BTV-­‐seronegative
 recipients.
 Ten
 of
 the
 19
 recipients
 were
 pregnant
 at
 60
 days
 when

pregnancy
  was
  terminated.
  The
  recipients
  remained
  BTV-­‐seronegative
  throughout.

(Bowen
 et
 al,
 Am
 J
 Vet
 Res
 1983;44:1625-­1628.)

Forty-­‐two
 normal
 embryos,
 9
 retarded
 embryos
 and
 the
 unfertilized
 ova
 collected
 from

infected
  donors
  were
  negative
  for
  BTV-­‐antigen
  (serotypes
  10,
  11,
  13,
  17)
  by

immunofluorescence.
 (Bowen
 et
 al,
 Am
 J
 Vet
 Res
 1983;44:1625-­1628.)

BTV
  was
  recovered
  from
  55%
  of
  flush
  fluids
  from
  non-­‐surgical
  flushes
  of
  viremic

donors,
 probably
 as
 a
 result
 of
 blood
 cell
 contamination.
 (Bowen
 et
 al,
 Am
 J
 Vet
 Res

1983;44:1625-­1628.)

A
 collaborative
 study
 by
 APHIS,
 ABADRL
 and
 MARC
 was
 done
 in
 1987-­‐88
 in
 the
 USA.
 To

avoid
  natural
  exposure
  to
  infection
  the
  animals
  were
  moved
  from
  Nebraska
  to

Wisconsin
  (a
  vector-­‐free
  area)
  in
  the
  vector
  season.
  Sixty
  heifers
  were
  infected

deliberately
 with
 BTV
 (serotype
 11)
 by
 bites
 of
 the
 vector
 and
 by
 inoculation
 with
 insect

2012 IETS research update 10