Bovine embryo–pathogen research

Ž . mended by the International Embryo Transfer Society Stringfellow, 1998 are simple, relatively inexpensive procedures that can be applied routinely in embryo production. Furthermore, embryo washing for health certification of in vivo-derived bovine embryos was validated in numerous studies that have been summarized in detail elsewhere Ž . Singh, 1987; Stringfellow et al., 1991; Anonymous, 1998a . In comparison, research on the epidemiology of in vivo-derived embryo production in small ruminants and swine and in vitro-derived embryo production in cattle has been less complete, and results indicate a greater tendency for association of pathogens with these embryos after Ž . artificial exposure Anonymous, 1998a; Guerin et al., 1997 . In this paper, results of embryo–pathogen research with in vivo-derived embryos of cattle, small ruminants and swine and with in vitro-derived embryos of cattle are summarized and compared, emphasizing potential for pathogen transmission under natural circumstances. In addi- tion, safe embryo handling practices and their application to multiple species and embryo production circumstances are discussed.

2. Bovine embryo–pathogen research

2.1. In ÕiÕo-deriÕed embryos of cattle Much of the initial research on interactions between bovine embryos and pathogens was designed to test a worst-case scenario through artificial exposure of zona pellucida- intact embryos to high concentrations of infectious agents. After exposure, embryos generally were washed 10 times to dilute unattached agent. Then embryos were ground or sonicated and assayed in vitro to determine if any infective agent remained associated with them after the washing procedure. Results of this research have been summarized Ž . see reviews: Anonymous, 1998a; Stringfellow and Givens, 1999 . Briefly, for eight of Ž nine viral pathogens akabane virus, bovine leukemia virus, bluetongue virus, bovine viral diarrhea virus, foot-and-mouth disease virus, bovine herpesvirus-1, bovine her- . pesvirus-4 and vesicular stomatitis virus that were used in these studies, infectious agent could not be isolated from embryos after washing or washing with trypsin . One Ž . virus rinderpest virus was stated in a preliminary report to associate with a small proportion of embryos after artificial exposure and washing. However, the finding was Ž . never confirmed. Also, for one Brucella abortus of six prokaryotic pathogens tested, washing was effective for removal while antibiotics in medium inactivated another Ž . Ž Haemophilus somnus . Finally, four of the six prokaryotes Mycoplasma boÕis, M. . boÕigenitalium, Mycobacterium paratuberculosis and Ureaplasma diÕersum could not be removed by washing after artificial exposure, but their association with embryos after natural exposure has never been shown. To determine the potential for natural exposure of embryos to pathogen or if pathogens would interact differently with embryos when exposure occurred in vivo, additional investigations were conducted in which zona pellucida-intact embryos and uterine recovery medium from artificially or naturally infected donor cows were collected and tested for infectious pathogen. To summarize the results, infectious agent was isolated from a proportion of samples of uterine recovery medium collected from Ž . Ž . cows infected with bovine leukemia virus 16 , bluetongue virus 40 , foot-and-mouth Ž . Ž . disease virus 68 and bovine herpesvirus-1 27 . However, after washing or washing with trypsin, embryos collected from cows artificially or naturally infected with bovine leukemia virus, bovine viral diarrhea virus, bluetongue virus, foot-and-mouth disease virus, bovine herpesvirus-1, rinderpest virus, Brucella abortus and Chlamydia Ž psittaci were all negative for infective agent see reviews: Anonymous, 1998a; Stringfel- . low and Givens, 1999 . Thus, pathogens were not associated with washed or trypsin washed embryos even when in vivo exposure to pathogen was confirmed by finding infective agent in the uterine recovery medium. Finally, research attempting to simulate natural circumstances was conducted. In these studies, embryos were collected from artificially or naturally infected cows, washed or washed with trypsin and transferred to uninfected recipients. Subsequently, recipients and offspring were monitored for disease. Such studies were conducted with donor cows that were infected with or seropositive to bovine leukemia virus, bluetongue virus, foot-and-mouth disease virus, bovine herpesvirus-1 and B. abortus. None of these Ž agents were transferred from donors to recipients or their embryo transfer offspring see . reviews: Anonymous, 1998a; Stringfellow and Givens, 1999 . Thus, based on assessment of general epidemiological factors associated with embryo Ž . transfer Stringfellow, 1985 and the results of specific investigations that are summa- rized above, it was clear that in vivo-derived, zona pellucida-intact bovine embryos are not likely to serve as vectors for transmission of disease if simple precautions such as washing or washing with trypsin are applied routinely in embryo processing. Research Ž . data is most complete i.e., evaluated using multiple experimental designs for the six pathogens listed in Table 1. It is noteworthy, that the Research Subcommittee of the Ž . International Embryo Transfer Society IETS ImportrExport Committee has concluded Table 1 Potential for transmission of pathogens through transfer of zona pellucida-intact, in vivo-derived, bovine embryos: summarized results of embryo–pathogen research Pathogen Potential for Pathogen associated with Recipientr natural exposure washed embryos calf infected to pathogen after transfer After artificial After natural from infected exposure exposure donor cows Bluetongue virus Possible No No No a a Bovine herpesvirus-1 Possible No No No Bovine leukemia virus Possible No No No Bovine viral diarrhea Possible No No No virus Foot-and-mouth disease Possible No No No virus Brucella abortus Unlikely No No No a Washing included treatment with trypsin. Washing procedures conformed to guidelines suggested by the Ž . International Embryo Transfer Society Stringfellow, 1998 . Information in this table summarized from reviews by: Anonymous, 1998a; and Stringfellow and Givens, 1999. Ž that five of the pathogens bluetongue, bovine herpesvirus-1, bovine leukemia virus, . foot-and-mouth disease virus and B. abortus in Table 1 are ‘‘disease agents for which sufficient evidence has accrued to show that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer’’ Ž . Anonymous, 1998b . The proper handling to which this statement refers is the use of standardized methods for washing and trypsin treatment of embryos that are described in Ž . the Manual of the International Embryo Transfer Society Stringfellow, 1998 . 2.2. In Õitro-deriÕed embryos of cattle There are reasonable concerns about transmission of pathogens with in vitro-pro- duced embryos of cattle, yet research on the topic has been limited compared to that done with in vivo-derived embryos. Topics of investigation have included exposure Ž potential i.e., possibility of introducing pathogens with raw materials used during in . vitro fertilization , the nature of embryo–pathogen associations and infectivity of embryo-associated pathogen. Ž . Ž . Bovine herpesvirus-1 BHV-1 and bovine viral diarrhea virus BVDV have been at the center of consideration because they are ever present in cattle populations and are Ž known to occur in serum and reproductive tissues Baker, 1995; Brock, 1998; Rossi et . al., 1980 . Cells and fluids from cattle infected with BHV-1 have been examined for virus. Cumulus–oocyte complexes, follicular fluid, granulosa cells, corpora lutea and uterine tubal cells all contained infective BHV-1 when collected from cattle acutely infected Ž . with the virus Bielanski and Dubuc, 1994; Guerin et al., 1989 . Further, the virus was isolated from in vitro-derived embryos produced with gametes and somatic cells from Ž . BHV-1-infected cattle Bielanski et al., 1998a , and when BHV-1 was artificially Ž introduced at various stages of in vitro embryo production systems Bielanski and . Dubuc, 1993; Bielanski et al., 1997 . Cells and fluids from cattle infected with BVDV have been examined for virus. Infectious BVDV was found in follicular fluid, cumulus–oocyte complexes and uterine Ž tubal cells from acutely infected cattle Bielanski and Dubuc, 1995; Bielanski et al., . 1998b and was isolated from ovarian tissue, follicular fluid, cumulus–oocyte com- plexes, granulosa cells, uterine tubal cells and semen from persistently infected cattle Ž . Bielanski and Loewen, 1994; Booth et al., 1995; Tsuboi and Imada, 1998 . Further, using indirect immunofluorescent assay of cryosections of ovaries from persistently infected heifers, BVDV antigen was found in 6 to 20 of oocytes in primordial, Ž . primary and secondary follicles Brownlie et al.,1997; Fray et al., 1998 . Thus, potential for introduction of BHV-1 and BVDV during in vitro production of embryos and the association of these viruses with developed embryos had been Ž demonstrated. Even more alarming was the discovery that embryo processing i.e., . Ž washing and trypsin treatment was ineffective for removing these viruses Bielanski . and Dubuc, 1993; Trachte et al., 1998 . Furthermore, embryo treatments were equally Ž . ineffective for removal of bluetongue virus Langston et al., 1999 , foot-and-mouth Ž . Ž disease virus Marquant-Le Guienne et al., 1998 and Leptospira Bielanski and . Surujballi, 1996 after artificial exposure. The question of how pathogens associate with in vitro-produced embryos was highlighted by the ineffectiveness of embryo processing procedures. In one study, leptospires were shown by electron microscopy to penetrate the zona pellucida and enter embryonic cells when embryos were artificially exposed in medium without antibiotics Ž . Bielanski and Surujballi, 1998 . However, the hazard of this occurring under normal circumstances appears to be low since this bacterium also was shown to be susceptible Ž to antibiotics commonly used in media for in vitro fertilization Bielanski and Surujballi, . 1996 . In experiments in which there was artificial exposure to viral pathogens, there was no evidence for penetration of the zona pellucida, but the viruses were obviously firmly attached. Ž . In a report by Vanroose et al. 1999 , mechanical entrapment by the zona pellucida was suggested as a mechanism for association of both BHV-1 and BVDV with the zona pellucida of in vitro-derived bovine embryos. They used confocal laser scanning microscopy to examine the location of fluorescent microspheres that were placed by positive pressure on the surface of the zona pellucida, and demonstrated that particles equivalent in size to BHV-1 and BVDV were able to penetrate through approximately 25 and 50 of the thickness of the zona pellucida, respectively. Thus, partial penetration and entrapment was possible while complete penetration and infection of embryonic cells was considered unlikely. Association of viral pathogens with the zona pellucida by entrapment had been hypothesized, but this report provided the first tangible evidence. Ž . A comparison of infective pathogens that associate presumably by entrapment or do not associate with either in vivo- or in vitro-derived, zona pellucida-intact, bovine embryos after exposure and washing or trypsin treatment is provided in Table 2. The trend is clear. Pathogens that were not associated with in vivo-derived embryos after Ž washing i.e., bluetongue virus, bovine herpesvirus-1, bovine viral diarrhea virus, and . foot-and-mouth disease virus did remain associated with in vitro-derived embryos Table 2 Comparison of infective pathogens associated with in vivo- or in vitro-derived, zona pellucida-intact, bovine embryos after in vitro or in vivo exposure to the pathogens and washing Pathogen In-vivo-derived embryos In-vitro-derived embryos After in vitro After in vivo After in vitro After in vivo exposure exposure exposure exposure Bluetongue virus No No Yes ND a a a a Bovine herpesvirus-1 No No Yes Yes a Bovine viral diarrhea virus No No Yes Yes Foot and Mouth Disease Virus No No Yes ND b b b,c Leptospira spp. ND No No No a Trypsin in washes. b Although infective agent was not isolated from washed embryos, some were positive by PCR assay. NDs Not determined. Washing procedures conformed to guidelines suggested by the International Embryo Ž . Transfer Society Stringfellow, 1998 . Information in this table summarized from reviews by Anonymous, 1998a; Bielanski, 1998; Stringfellow and Givens, 1999; and from Bielanski et al., 1998c; Langston et al., 1999; Marquant-Le Guienne et al., 1998; and Trachte et al., 1998. c Antibiotics in in vitro culture medium. despite processing according to procedures recommended by the International Embryo Ž . Transfer Society for in vivo-derived embryos Stringfellow, 1998 . A summary of available information relative to the possibility of transmission of these pathogens with transferred, in vitro-derived embryos is provided in Table 3. The known potential for Ž natural exposure to at least two of these pathogens bovine herpesvirus-1 and bovine . viral diarrhea virus and the relative ineffectiveness of embryo processing procedures Ž . washing and trypsin treatment create legitimate concerns that they might be transmit- ted with exposed embryos. However, it has not been determined that the quantity of infectious virus associated with these embryos would constitute an infective dose for susceptible recipients via the intrauterine route. Of course, risk assessment cannot be complete without establishing whether or not the amount of embryo-associated pathogen constitutes an infective dose. The question of infective dose has been addressed to some degree with bovine viral diarrhea virus. Exposed embryos were shown to be infective when they were washed, sonicated and Ž . injected intravenously into susceptible cattle Bielanski and Jordan, 1996 . However, Ž . under natural circumstances embryos would be intact not sonicated and would be delivered individually into recipients via the intrauterine route. Apparently contrasting Ž . results were presented in a report by Givens et al. 1999a . They discovered that the bovine viral diarrhea virus associated with single, washed, unsonicated, in vitro-derived embryos did not infect susceptible uterine tubal cells co-cultured with them for two days. In a subsequent study, the same investigators examined limited quantity and loss of infectivity of embryo-associated bovine viral diarrhea virus and antiviral influence of the embryo as possible explanations for failure of individual, developing, virus-exposed Ž . embryos to infect uterine tubal cells in co-cultures Givens et al., 1999b . Their results indicated that each of the three factors was partially responsible for preventing infection of the uterine tubal cells, but the relative importance of each factor could not be determined. These results are interesting and seem to indicate that an infective dose of bovine viral diarrhea virus might not be associated with exposed, transferred embryos. However, final resolution of the question can be accomplished only by transfer of Table 3 Potential for transmission of pathogens through transfer of zona pellucida-intact, in vitro-derived, bovine embryos: Summarized results of embryo-pathogen research Pathogen Potential for Pathogen associated with Recipientr natural exposure washed embryos calf infected to pathogen after transfer After artificial After natural from infected exposure exposure donor cows Bluetongue virus Uncertain Yes ND ND Bovine herpesvirus-1 Possible Yes Yes ND Bovine viral diarrhea virus Possible Yes Yes ND Foot-and-mouth disease virus Uncertain Yes ND ND NDs Not determined. Washing procedures conformed to guidelines suggested by the International Embryo Ž . Ž . Transfer Society Stringfellow, 1998 . Information in this table summarized from Bielanski 1998 , Langston Ž . Ž . Ž . et al. 1999 , Marquant-Lequienne et al. 1998 , and Trachte et al. 1998 . exposed embryos to susceptible recipients and monitoring recipients and offspring for signs of infection.

3. Embryo–pathogen research in small ruminants and swine