111 RISK OF CHLAMYDIA ABORTUS TRANSMISSION VIA EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS

Abstract

Chlamydia abortus (C. abortus) in cattle has been reported sporadically throughout the world and is implicated in respiratory, ocular, and reproductive disease as abortion, infertility, chronic mastitis, vaginal discharge, and endometritis. In addition, C. abortus presents a zoonotic risk exposure of pregnant women to infected animal and can lead to severe septicaemia in the mother, resulting in spontaneous abortion or stillbirth of the fetus. To investigate the risk of C. abortus transmission via bovine embryo transfer, our study aims to determine whether the embryonic ZP of in vitro-produced embryos protects early embryo cells against C. abortus infection and whether the bacteria adhere to or infect the cells of early bovine embryos (ZP-free) after in vitro infection. We also evaluated the efficacy of the washing procedure recommended by the IETS to decontaminate bovine embryos exposed to C. abortus in vitro. Ninety (8 to 16 cells) bovine embryos, produced in vitro, were randomly divided into 10 batches. Eight batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4.8 × 10⁷ Chlamydia/mL of AB7 strain (ANSES, Maisons-Alfort, France). After incubation for 18 h at 37°C in an atmosphere of 5% CO₂, the embryos were washed in batches in 10 successive baths of a PBS and 5% FCS solution without trypsin nor antibiotics in accordance with IETS guidelines. In parallel, 2 batches of 5 embryos (1 ZP-intact and 1 ZP-free) were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13 000 × g. The embryos and wash pellets were tested using RT-PCR. Chlamydia abortus DNA was found in all ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the tenth wash fluid for 1 batch (1/4) of ZP-intact infected embryos and in 3 batches (3/4) of ZP-free infected embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. These results demonstrate that C. abortus adheres to or penetrates the ZP as well as the early embryonic cells of in vitro-produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients or their offspring. Nevertheless, the finding of C. abortus DNA by RT-PCR did not imply that the bacteria found is still infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. abortus would eliminate the bacteria from the ZP.