90 Risk of Coxiella burnetii Transmission via Embryo Transfer Using In Vitro Early Caprine Embryos

Abstract

Previous experiments using in vitro infection have shown that Coxiella burnetii has a strong tendency to adhere to the zona pellucida (ZP) of in vivo-derived goat embryos, and the washing procedure recommended by the International Embryo Technology Society (IETS) for bovine embryos failed to remove it (Alsaleh et al. 2013 Theriogenology 80, 571-575). The aim of this study was, for in vitro-produced caprine embryos infected in vitro, to (1) evaluate the ability of C. burnetii to adhere to intact ZP, (2) test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii, and (3) determine, by confocal microscopy, the bacteria location. One hundred ZP-intact caprine embryos, produced in vitro, at the 8- to 16-cell stage were randomly allocated into 11 batches of 8 to 9 embryos. Nine batches were incubated for 18 h with 10⁹ Coxiella/mL of CbB1 strain (ISP, INRA, Val de Loire, France). The embryos were then recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, 2 batches of embryos were subjected to similar procedures but without exposure to C. burnetii to serve as the control group. One of the 9 batches of infected embryos and 1 of the 2 non-infected control batches were used to perform immunolabelling to localise the bacteria. Coxiella burnetii DNA was detected by conventional PCR in all 8 batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryos of the control group. The first 5 washing media of the infected groups were consistently positive and Coxiella DNA was detected up to the tenth wash in 2 batches. After immunolabelling, the observation of embryos under confocal microscopy allowed us to localise C. burnetti on the external part of the ZP without deep penetration. The presence of C. burnetii was seen on the surface of the ZP, with bacterial loads differing from one embryo to another in the same batch. This study clearly demonstrates that C. burnetii, after in vitro infection at 10⁹ Coxiella mL⁻¹, stick strongly to the external part of the ZP of in vitro-produced early caprine embryos without profound penetration. The 10-washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria in caprine embryos. Nevertheless, the finding of C. burnetii DNA by conventional PCR does not imply that the bacteria found remain infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of caprine embryos infected by C. burnetii would eliminate or inactivate the bacteria from the ZP of in vitro-produced goat embryos.