Pellerin J L, Alsaleh A, Mermillod P, Souza-Fabjan J M G, Rodolakis A, Rousset E, Dubreil L, Bruyas J F, Roux C, Fieni F
LUNAM Université, Oniris, Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering, CS 44706, Nantes, F-44307, France; UPSP 5301 DGER, France, 44307 Nantes Cedex 03, France.
LUNAM Université, Oniris, Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering, CS 44706, Nantes, F-44307, France; UPSP 5301 DGER, France, 44307 Nantes Cedex 03, France.
Theriogenology. 2018 Jan 15;106:259-264. doi: 10.1016/j.theriogenology.2017.10.033. Epub 2017 Oct 26.
Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii. One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 10Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batches were separated to perform immunolabeling to locate the bacteria. C. burnetii DNA was detected by C-PCR in all eight batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryo control batch. The first five washing media of the infected group were consistently found to be positive and Coxiella DNA was detected in the wash bath up to the 10th wash for two batches. After immunolabeling, the observation of embryos under confocal microscopy allowed C. burnetti to be found on the external part of the zona pellucida without deep penetration. This study clearly demonstrates that C. burnetii, after in vitro infection at 10Coxiella/ml, stick strongly to the external part of the zona pellucida of in vitro produced caprine embryos without deap penetration and that the 10 washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria from in vitro-produced goat embryo.
先前的研究表明，在体内来源的山羊胚胎受到感染后，伯氏考克斯体（C. burnetii）表现出强烈的附着于透明带（ZP）的倾向。为了调查通过体外生产的山羊胚胎进行胚胎移植传播伯氏考克斯体的风险，本研究的目的是：（i）评估伯氏考克斯体附着于体外生产的山羊胚胎完整透明带的能力，并通过共聚焦显微镜确定细菌的位置；（ii）测试国际胚胎移植协会（IETS）推荐的牛胚胎洗涤规则对消除伯氏考克斯体的效果。100个体外生产的处于8至16细胞阶段的完整透明带山羊胚胎被随机分为11组，每组8至9个胚胎。9组胚胎与每毫升含10个伯氏考克斯体的CbB1菌株（IASP，法国国家农业研究院图尔研究中心）一起孵育18小时。然后回收胚胎，并按照IETS指南在10个连续的浴中分批洗涤。同时，两组胚胎接受类似的程序，但不接触伯氏考克斯体，作为对照组。9组感染胚胎中的一组和2组未感染对照胚胎中的一组被分离出来进行免疫标记以定位细菌。在连续10次洗涤后，通过C-PCR在所有8组感染胚胎中均检测到伯氏考克斯体DNA。然而，在胚胎对照组中未检测到细菌DNA。感染组的前五个洗涤培养基一直呈阳性，并且在两批胚胎的第10次洗涤前的洗涤浴中均检测到考克斯体DNA。免疫标记后，在共聚焦显微镜下观察胚胎发现，伯氏考克斯体位于透明带外部，未深入穿透。本研究清楚地表明，在每毫升含10个伯氏考克斯体的体外感染后，伯氏考克斯体强烈附着于体外生产的山羊胚胎透明带的外部，未深入穿透，并且IETS推荐的用于消除牛胚胎病原体的10次洗涤方案无法从体外生产的山羊胚胎中消除这些细菌。
