Health and Safety Advisory Committee Research Update PDF
Preview Health and Safety Advisory Committee Research Update
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
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