Embryo–pathogen research in small ruminants and swine

exposed embryos to susceptible recipients and monitoring recipients and offspring for signs of infection.

3. Embryo–pathogen research in small ruminants and swine

Information on embryo–pathogen interactions using in vitro-derived embryos of small ruminants and swine is insufficient to discuss here. Available information on research related to in vivo-derived embryo pathogen interactions in swine, sheep and Ž goats has been reviewed in detail elsewhere Stringfellow et al., 1991; Anonymous, . 1998a; Thibier and Guerin, 1999 . A brief synopsis from these reviews and selected individual reports is provided in this paper. 3.1. Swine Ž Swine embryos were artificially exposed to seven different pathogens African swine fever virus, foot-and-mouth disease virus, hog cholera virus, porcine parvovirus, pseu- . dorabies virus, swine vesicular disease virus, and vesicular stomatitis virus , washed as Ž . recommended Stringfellow, 1998 and assayed for infective virus with the result that at Ž least some embryos were always positive for each of the pathogens reviewed in . Stringfellow et al., 1991; Anonymous, 1998a . Further, in one study, a nucleic acid probe was used to detect replicating porcine parvovirus, indicating that the virus had Ž . crossed the zona pellucida and infected embryonic cells Bane et al., 1990 . Since all pathogens evaluated by artificial exposure remained associated with washed porcine embryos, it was especially important to evaluate the health status of recipients and offspring after transfer of washed or trypsin-treated, zona pellucida-intact embryos collected from infected or seropositive donors. In such experiments with embryo donors Ž . that were infected with hog cholera virus Anonymous, 1998a , porcine reproductive and Ž . respiratory syndrome virus Randall et al., 1999 and swine vesicular disease virus Ž . Singh et al., 1987 , recipients and offspring remained disease-free. In such studies with Ž donors that were pseudorabies virus-infectedrseropositive summarized in Stringfellow . et al., 1991 , when embryos were trypsin treated, there was no evidence of disease in recipients and offspring. Thus, it can be concluded that demonstrating association of a pathogen with swine embryos after artificial exposure and washing is not necessarily accompanied by a high risk that embryos collected from infected donors will infect recipients or offspring after transfer. Ž . The report by Bane et al. 1990 that porcine parvovirus penetrated the intact zona pellucida and replicated in embryonic cells after artificial exposure remains a concern. Since this is the only report of a viral pathogen penetrating the zona pellucida of an embryo from any livestock species, it seems appropriate that the experiment be confirmed by duplication. 3.2. Goats Specific information on epidemiology of embryo production in goats is very limited Ž . see review by Thibier and Guerin, 1999 . There is one reference to an in vitro-exposure Ž . study testing the efficacy of washing caprine embryos Thibier, 1990 . In this study, Ž 3 . embryos collected from disease-free does were exposed in vitro to low 10 , medium Ž 5 . Ž 7 . 10 and high 10 concentrations of M. mycoides mycoides, washed using recom- Ž . mended procedures Stringfellow, 1998 and assayed for the presence of the pathogen. Mycoplasmas were isolated from all washed embryos that had been exposed to medium or high concentrations of the organism. There have been three reports of studies in which caprine embryos were collected from donors that were naturally or artificially exposed to pathogen and transferred to Ž . disease-free recipients. Chemineau et al. 1986 collected embryos from does in a flock in which approximately half of the animals were seropositive to bluetongue virus. The embryos were washed 10 times, cryopreserved and later transferred to disease-free Ž . Ž . recipients. None of the recipients n s 19 or offspring n s 19 developed antibody to Ž . bluetongue virus. Wolfe et al. 1987 collected embryos from does that were seroposi- tive to caprine arthritis-encephalitis virus. The embryos were washed only three times Ž . before transfer to negative recipients. However, the recipients n s 8 and offspring Ž . Ž . n s 1 remained seronegative. Finally, Foster et al 1999 collected embryos from does Ž . that had been artificially infected with bovine spongiform encephalopathy BSE . Embryos were washed 10 times and transferred to BSE-free recipients. Again, recipients Ž . Ž . n s 22 and offspring n s 37 remained free of signs of disease and were negative by all analyses. Presumably, the latter study represented the use of a caprine model to study possible transmission of BSE via embryo transfer, since BSE is generally considered a disease of cattle and not of goats. Although the work with caprine embryos has been limited, one trend is similar to that found with both bovine and porcine embryos. When properly washed embryos from pathogen-exposed donors are transferred to disease-free recipients, both offspring and recipients remained free of disease. 3.3. Sheep Ž Zona pellucida-intact ovine embryos were exposed in vitro to one viral bovine viral . Ž . diarrhea virus and three bacterial pathogens B. abortus, B. oÕis, Campylobacter fetus . Then embryos were washed and examined in vitro for infectious agent. The combination of washing and treatment with anti-bovine viral diarrhea virus antibody was effective for Ž . removalrinactivation of bovine viral diarrhea virus Evermann et al., 1981 . Further, Ž . washing was effective for removal of Campylobacter fetus Guerin et al., 1988 , but in the absence of antibiotics, washing was unreliable for the removal of B. abortus Ž . Riddell et al., 1989 . Also, neither washing nor treatment with antibiotics was effective Ž . for the removal of B. oÕis Wolfe et al., 1988; Guerin et al., 1992 , and when embryos Ž . exposed in vitro to B. oÕis were washed no antibiotics and transferred, four of seven Ž . recipients seroconverted Riddell et al., 1990 . Possible transmission of bluetongue virus, C. psittaci, sheep pulmonary adenomatosis and scrapie under natural circumstances were evaluated by the collection of embryos from affected ewes and transferred to recipients. Ž . Ž . In a study by Hare et al. 1988 , embryos n s 49 were collected from ewes that had been artificially infected with bluetongue virus. Subsequently, the embryos were washed using procedures that have since been adopted by the International Embryo Transfer Ž . Ž . Society Stringfellow, 1998 and transferred to bluetongue virus-free recipients n s 27 . Ž . None of the recipients or offspring n s 16 seroconverted. However, in a similar study Ž . Ž . by Gilbert et al. 1987 , 20 embryos were collected from viremic donors n s 8 and Ž . transferred to susceptible recipients n s 15 . Viremia and seroconversion occurred in 2 of the 15 recipients, although one of these recipients gave birth to a lamb that was seronegative and virus-negative. Unfortunately, there was no mention of an attempt to wash the embryos, raising concern that embryos were not properly washed and making accurate interpretation of the results difficult. The feasibility of using embryo transfer to break the cycles of infection for C. psittaci Ž . and sheep pulmonary adenomatosis SPA was demonstrated in two other studies. Ž . Ž . Williams et al. 1998 reported that properly washed embryos n s 12 from ewes Ž . n s 3 artificially infected with C. psittaci did not result in infection of recipients Ž . Ž . n s 7 or their offspring. Also, Parker et al. 1998 collected 215 embryos from 76 Ž . ewes in a flock endemic for SPA, washed them as recommended Stringfellow, 1998 and transferred them to 131 negative recipients. None of the recipients or 38 offspring that were from ewes confirmed to be SPA-affected showed signs of disease. Table 4 Status of recipientsroffspring after transfer of properly washed, zona pellucida-intact, in vivo-derived embryos from infected or seropositive donors Donor Species Pathogen Seroconversionrinfection of recipientroffspring? Cattle Bluetongue virus No a Cattle Bovine herpesevirus-1 No Cattle Bovine leukemia virus No Cattle Bovine viral diarrhea virus No Cattle Foot-and-mouth disease virus No Cattle Brucella abortus No b Cattle Bovine spongiform encephalopathy No c Swine Hog cholera virus No Swine Porcine reproductive and respiratory syndrome virus No a Swine Pseudorabies virus No Swine Swine vesicular disease virus No Sheep Bluetongue virus No c Sheep Maedi-Visna virus No Sheep Sheep pulmonary adenomatosis No Ž . Sheep Scrapie YesrNo disputed Sheep Chlamydia psittaci No Goat Bluetongue virus No Goat Caprine arthritis–encephalitis virus No Goat Bovine spongiform encephalopathy No a Washing included use of trypsin. b Longterm study still in progress. c Results reported as unpublished. Washing procedures conformed to guidelines suggested by the Interna- Ž . tional Embryo Transfer Society Stringfellow, 1998 . Information in this table from reviews by: Anonymous, 1998a; Stringfellow et al., 1991; Stringfellow and Givens, 1999; Wrathall and Sutmoller, 1998; and from Foote et al., 1993; Foster et al., 1996, 1999; Singh et al., 1987; Randall et al., 1999; and Woodall et al., 1993. The potential for transmission of scrapie via embryos has been the topic of investiga- Ž . tion by two research teams. Foote et al. 1993 collected embryos from scrapie-inoc- ulated ewes, washed them three times without regard to condition of the zona pellucida and transferred them fresh to recipients in a scrapie-free flock. None of the recipients, including 129 that lambed, or offspring, including 56 that survived to 5 years of age, developed signs or lesions indicative of scrapie. However, conflicting results were Ž . presented by Foster et al. 1992 . In this study, 37 embryos were transferred from donor ewes artificially infected with scrapie into 16 recipients with birth of 26 lambs resulting. They described this study as a worst-case circumstance in which embryos were not washed. Six of the lambs developed scrapie at 2 to 2.5 years of age. Then, in a Ž . subsequent study Foster et al., 1996 , the same investigators reported that properly washed embryos from infected ewes resulted in lambs that later developed scrapie. However, validity of their results has to be questioned, since the flock of origin of recipients had a low incidence of natural scrapie, and they reported that the offspring Ž . Ž . two of four from transfer of susceptible sAsA , negative-control, washed embryos also developed scrapie. It appears that the trend established through research in cattle, swine and goats has continued in research with sheep. When embryos from pathogen exposed donors were Ž . properly treated Stringfellow, 1998 before transfer to disease-free recipients, both Ž . offspring and recipients remained free of disease Table 4 . The one possible exception Ž . is found in the report by Foster et al. 1996 . However, in their study, recipients came from a flock with a low incidence of natural scrapie, and some negative control animals developed scrapie. Thus, there was a realistic possibility that scrapie was not actually transmitted with washed embryos.

4. Discussion about safe embryo handling procedures