Embryo transfer as a means of controlling the transmission of viral infections. XV. Failure to transmit bluetongue virus through the transfer of embryos from viremic sheep donors

The first experiment involved in vitro exposure of clean embryos to bluetongue virus (BTV) while three subsequent experiments involved the collection of embryos from BTV-infected donor ewes and their transfer to disease-free recipients. In Experiment I, 22 embryos/ova were exposed to BTV type 11 (BTV-11) for 1 h, washed 10 times in PBS and assayed in pairs for BTV. All 11 samples were positive for BTV in the 11-d-old embryonated chicken egg (ECE) assay system and 5/11 samples were positive in baby hamster kidney-21 (BHK-21) cells. In Experiment II, 5 donors were infected with BTV type 10 (BTV-10). All embryos were washed 10 times prior to assay or transfer. Thirty-three embryos/ova were assayed in groups of 2 or 3 and none yielded virus in ECE. Two BTV-seronegative recipients each received 6 embryos and a total of 3 lambs free of BTV antibodies were delivered. In Experiments III and IV, a total of 9 donors were infected with BTV-11. All embryos were washed 10 times prior to assay or transfer. Seventy-four embryos/ova were assayed in groups of 2 or 3 and none yielded virus in ECE, while for each experiment, 6 embryos were transferred into 2 BTV-seronegative recipients. The four recipients and their 3 lambs and 2 aborted fetuses were also seronegative for BTV.     

Embryo transfer as a means of controlling the transmission of viral infections. XIII. Failure to transmit foot-and-mouth disease virus through the transfer of embryos from viremic donors

A total of 436 embryos/unfertilized ova was collected from 30 foot-and-mouth disease (FMD) viremic cattle; 106 of these embryos/ova were from eight donors that had FMD virus in their reproductive tracts. The 436 embryos/ova were washed and then either assayed in cell culture or intradermally in steer tongues or transferred to recipients. Foot-and-mouth infectivity was not found to be associated with any of the embryos/ova assayed in cell culture or intradermally. The 149 embryos transferred produced two abortions, five sets of twins born prematurely, and 15 normal calves. All of the recipients and all of the calves remained FMD-seronegative.     

Embryo transfer as a means of controlling viral infections. VI. Bluetongue virus-free calves from infectious semen

Four Holstein heifers were superovulated and inseminated with infectious semen from a bull experimentally infected with type 17 bluetongue virus (BTV). A total of 20 embryos were collected at donor slaughter and transferred to 16 recipients. Ten recipients became pregnant of which one subsequently aborted, one gave birth to twins which died at birth, one was killed at term because of dystocia, and 7 gave birth to live calves one of which died perinatally. All animals were tested for BTV antibodies at the time of slaughter which was at least 30 days post partum for surviving heifers and calves. Two of the four donor heifers were retrospectively determined to have been infected by the semen (viremia demonstrated) and their embryos accounted for 9 of the 10 pregnancies including the six surviving calves. None of the recipients or calves developed BTV antibody by the termination of the experiment. This study suggests that BTV-free calves can be readily obtained from the use of BTV-positive semen.     

Embryo transfer as a means of controlling the transmission of viral infections. VIII. Failure to detect foot-and-mouth disease viral infectivity associated with embryos collected from infected donor cattle

Foot-and-mouth disease (FMD) viral infectivity detectable in cell cultures or by animal inoculation was not found to be associated with any of 48 washed zona pellucida-intact (ZPI) embryos collected from 8 cattle during the acute stages of disease. Similarly, infectivity was not found to be associated with any of 42 washed ZPI embryos collected from 3 cattle 21 d after infection with FMD.     

Embryo transfer as a means of controlling the transmission of viral infections. I. The in vitro exposure of preimplantation bovine embryos to akabane, bluetongue and bovine viral diarrhea viruses

As part of a program to study the feasibility of using embryo transfer to control disease, initial experiments were undertaken to determine the virus susceptibility of early embryos. Two hundred and ninety-three preimplantation bovine embryos (16-cell to blastocyst stage) were exposed to either akabane virus (AV), bluetongue virus (BTV) or bovine viral diarrhea virus (BVDV). Two hundred and thirty-seven of these embryos were then cultured for 24-48 hours in order to determine whether the virus had any effect on embryonic development and to allow viral replication to occur. No infectious virus was isolated from any of the embryos and the in vitro development of virus exposed embryos proceeded normally. In addition, twenty-nine eggs/embryos isolated from donors that were seropositive to BVDV were found to be uninfected with this virus.     

Embryo transfer as a means of controlling the transmission of viral infections. XI. The in vitro exposure of bovine and porcine embryos to vesicular stomatitis virus

Infectious virus was isolated from both porcine and bovine zona pellucida-intact embryos that had been exposed to the Indiana strain of vesicular stomatitis virus (VSV) and then washed. The amount of virus isolated from embryos depended on their initial exposure level. Porcine embryos always retained more virus than bovine embryos. When embryos were cultured for 24 h after viral exposure and washing, the number of embryos carrying VSV and the amount of virus on each of the embryos was reduced. Trypsin (0.25%) was also found to be effective in inactivating/removing the VSV from embryos, suggesting that most, if not all, of the virus was bound to the zona pellucida.     

Embryo transfer as a means of controlling the transmission of viral infections. VII. The in vitro exposure of bovine and porcine embryos to foot-and-mouth disease virus

When 169 zona pellucida-intact bovine embryos were exposed to 10(6) pfu/ml of foot-and-mouth disease virus and then washed, no infectious virus was detected on any of the embryos. FMD viral infectivity was found, however, in association with 14 of 42 hatched (zona pellucida-free) bovine embryos and in a small number of zona pellucida-intact porcine embryos. The porcine embryos were assayed individually and in groups of 8 embryos. Four of the 124 individual embryos and 2 of the 9 groups of embryos carried the infectious virus.     

Embryo transfer as a means of controlling the transmission of viral infections. X. The in vivo exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus

Two experiments involving the transfer of embryos from donors infected with swine vesicular disease virus (SVDV) to "clean" recipients were carried out. In Experiment 1, 47 embryos were collected from 4 SVDV-infected donors and transferred to 2 recipients that subsequently produced 10 piglets. All of the recipients and piglets remained seronegative for SVDV. In addition to the transfers, 10 embryos and 58 unfertilized eggs from the infected donors were assayed in vitro and found to be negative for SVDV infectivity. A fifth donor was also inoculated with SVDV in this experiment, but it could not be demonstrated that infection had occurred. This SVDV-exposed donor provided two embryos for transfer and one embryo and two unfertilized eggs for in vitro assay. In Experiment 2, 158 embryos from 9 infected donors were transferred to 7 recipients, resulting in 12 piglets. A total of 7 embryos and 37 unfertilized eggs were assayed in vitro. The recipients, piglets, and embryos/eggs were all negative for SVDV infectivity. Although a final conclusion on the safety of using embryo transfer for the control of swine vesicular disease (SVD) is not possible, the results obtained justify additional studies.     

Embryo transfer as a means of controlling the transmission of viral infections. IX. The in vitro exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus

When zona pellucida-intact porcine embryos were exposed to 10(7) plaque-forming units (pfu)/ml of swine vesicular disease virus (SVDV) and then washed, infectious virus could be isolated from all of the embryos. Culturing the embryos for 24 or 48 h or treating the embryos with pronase, trypsin, or antiserum after virus exposure and washing reduced the number of embryos carrying virus and lessened the amount of virus on each of the embryos. None of the treatments, however, was capable of disinfecting every embryo.     

Embryo transfer as a means of controlling the transmission of viral infections: V. the in vitro exposure of zona pellucida-intact porcine embryos to African swine fever virus

African swine fever virus (ASFV) was detected on or in zona pellucida-intact porcine embryos that had been exposed to 10^6.6 hemadsorption dose 50%/ml (HAdD₅₀/ml) of ASFV for 18 hours, washed and then cultured. Ninety-five percent of the embryos retained infectious virus after washing. Treating the embryos with papain, EDTA or ficin had no effect on the retained virus, whereas treating them with trypsin or pronase reduced the number of embryos carrying detectable virus (30% instead of 95%) and lowered the amount of virus on the embryos. It has not yet been determined whether ASFV enters the embryonic cells but the evidence suggests that most of the virus, and possibly all of it, is bound to the zona pellucida.     

Embryo transfer from donor cattle persistently infected with bovine viral diarrhea virus

This study was done to examine the reproductive efficiency of embryo transfer donors that were persistently infected with bovine viral diarrhea virus (BVDV) and to determine the potential for vertical or horizontal transmission of BVDV during embryo transfer from persistently infected donors. The reproductive inefficiency of 7 different persistently infected donors was evident by consistent failure at superovulation and/or fertilization. Washing of embryos according to the reccommendations of the International Embryo Transfer Society (IETS) prevented the adherence of BVDV to embryos and to unfertile and degenerated ova, as determined by virus isolation and polymerase chain reaction (PCR) assay. In addition, a normal, BVDV antibody seronegative and BVDV-negative calf was born following transfer from a PI donor to a seronegative recipient.     

Evaluation of housing as a means to protect cattle from Culicoides biting midges, the vectors of bluetongue virus

The housing of animals at night was investigated as a possible means of protecting them from attack by Culicoides biting midges (Diptera: Ceratopogonidae), the vectors of bluetongue. Light-trap catches of Culicoides were compared inside and outside animal housing, in the presence and absence of cattle. A three-replicate, 4 × 4 Latin square design was used at four farms in Bala, north Wales, over 12 nights in May and June 2007, and the experiment repeated in October. In the two studies, respectively, >70 000 and >4500 Culicoides were trapped, of which 93% and 86%, respectively, were of the Culicoides obsoletus group. Across the four farms, in May and June, the presence of cattle increased catches of C. obsoletus by 2.3 times, and outside traps caught 6.5 times more insects than inside traps. Similar patterns were apparent in October, but the difference between inside and outside catches was reduced. Catches were strongly correlated with minimum temperature and maximum wind speed and these two variables explained a large amount of night-to-night variation in catch. Outside catches were reduced, to a greater extent than inside catches, by colder minimum temperatures and higher maximum wind speeds. These conditions occur more frequently in October than in May and June, thereby suppressing outside catches more than inside catches, and reducing the apparent degree of exophily of C. obsoletus in autumn. The results suggest that the risk of animals receiving bites from C. obsoletus is reduced by housing at both times of year and the benefit would be greatest on warm, still nights when outside catches are at their greatest.     

The transmission of bluetongue virus by embryo transfer in sheep

The susceptibility of sheep to intrauterine infection with bluetongue virus (BTV) was established by introducing 10(4) plaque-forming units of BTV type 10 into the uterine lumen of two seronegative ewes in a simulated embryo transfer operation. Both ewes became viraemic and underwent seroconversion to BTV. Embryos recovered from seronegative superovulated donor ewes were incubated in vitro for 8 h with BTV type 10. After incubation the embryos were thoroughly rinsed by repeated transfer to sterile culture medium, and 12 of these embryos were then transferred to the uterus or oviduct of seronegative, synchronized recipients. Viraemia and seroconversion were detected in nine recipient ewes. Embryos recovered from eight viraemic ewes were transferred to 15 seronegative, synchronized recipients. Viraemia and seroconversion were detected in 2 of the recipients, both of which also became pregnant. A lamb born to a ewe becoming infected at the time of embryo transfer was clinically normal, and no evidence of BTV infection was obtained at postmortem examination of the lamb after slaughter.     

Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue transmission

In order to import non-seasonal Creole goats from the Carribean to Europe for an experimental purpose, thirty Creole goats were treated with 10 mg of FSH; embryos were collected at slaughter, washed and deep frozen. After rapid thawing, they were reimplanted surgically into European dairy goats. Twenty-four females ovulated but only 17 of the ovulating females had functional corpora lutea (CL) at collection. Ovulation rate (CL goat ) and recovery rate (embryo CL ) were 13.8 and 78% for females with functional CL. Of 191 embryonic structures collected, 79% were considered suitable for deep freezing: 23% were young blastocysts, 47% were expanded blastocysts, and 30% were zona-pellucida (zp)-free and zp-damaged embryos. Seventy-eight embryos were thawed and 63 were reimplanted. Sixty-eight percent of the recipient females delivered 19 kids. The percentage of kids born relative to good-quality re-implanted embryos was higher for zp-free embryos (64%) than for young and expanded blastocyts (36%). Forty-seven percent of the donor females had strong positive serological reactions for bluetongue virus antibodies against serotypes 6 and 14. However, no recipient goats or newborn kids were positive. Virus isolation attempts on the collection media and last embryo washes were negative.     

An attempt at embryo transfer as a means of controlling Bordetella bronchiseptica infection in the rabbit

Embryo transfer was attempted in order to control disease in rabbits. Embryos were collected by flushing of the oviducts of donor rabbits on Day 2 of gestation, into small tubes containing the medium, transported within the body warmth of the person carrying the tubes and transferred into the oviducts of SPF pseudopregnant recipients. The time between embryo collection and transfer was 7-8 hours. Ten of 56 embryos derived from Bordetella bronchiseptica infected animals developed into newborns. As a result of bacteriological examination of intranasal exudate in six weanlings, no pathogens were detected. We suggest that embryo transfer is an effective and simple alternative to caesarean operation in Bordetella bronchiseptica infected rabbits.     

Failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients or their offspring

Sixty heifers were infected with bluetongue virus (BTV) by the bites of the vector and by inoculation with insect origin virus. During the acute and convalescent stages of the infection, embryos were collected nonsurgically from these animals and washed according to the recommendations of the International Embryo Transfer Society (1). No BTV was isolated from 77 of these embryos when they were inoculated onto cell culture and into embryonating chicken eggs. There was no evidence of lateral BTV transmission when 231 of these embryos were transferred into susceptible recipients, nor was there evidence of vertical BTV transmission to the 88 calves resulting from these transfers. Another six donors that were assumed to have recovered from a natural infection of BTV, were added to the study to increase the probability of obtaining embryos from a persistently infected BTV carrier. However, it was determined later that these animals had not been infected with BTV but with the closely-related epizootic hemorrhagic disease virus (EHDV). Embryos were collected from these donors and washed as above. Neither BTV nor EHDV was isolated from 26 of these embryos by the inoculation of cell culture and embryonating chicken eggs. There was no evidence of lateral BTV or EHDV transmission to recipients of 15 of these embryos or of vertical BTV or EHDV transmission to the resulting 7 calves. However, two recipients of embryos from one of these donors developed antibodies to BTV 6 to 9 months after transfer. Passive antibodies to BTV were also detected in their calves. There is good evidence that these two recipients acquired BTV from natural exposure to infected insect vectors and not from the transferred embryos.