SALUVET, Animal Health Department, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
Department of Anatomy, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173, Hannover, Germany.
Parasit Vectors. 2017 Oct 10;10(1):463. doi: 10.1186/s13071-017-2409-9.
Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the placental barrier and placental damage is crucial in the pathogenesis of abortion. Bovine trophoblast and caruncular cell layers are key cellular components in the maternal-foetal interface in placentomes, playing a fundamental role in placental functionality.
We studied tachyzoite adhesion, invasion, proliferation and egress of high- (Nc-Spain7) and low- (Nc-Spain1H) virulence N. caninum isolates in established cultures of bovine caruncular epithelial (BCEC-1) and trophoblast (F3) cells. The parasite invasion rate (pInvR) and the cell infection rate (cInfR) were determined by immunostaining plaque assay at different time points and multiplicities of infection (MOIs), respectively. In addition, tachyzoite growth kinetics were investigated using real-time PCR (qPCR) analysis and immunostaining plaque assay at different times.
Neospora caninum invaded and proliferated in both cell lines. The pInvR was higher in F3 compared to BCEC-1 cells for the Nc-Spain7 isolate (P < 0.05), and higher for the Nc-Spain7 than the Nc-Spain1H in F3 cells (P < 0.01). The cInfR was also higher in F3 cells than in BCEC-1 cells for both isolates (P < 0.0001), and the cInfR for the Nc-Spain7 isolate was higher than for the Nc-Spain1H isolate in both cell lines (P < 0.05). Tachyzoite growth kinetics showed tachyzoite exponential growth until egress at 58 hpi for both isolates in F3, whereas Nc-Spain1H showed a non-exponential growth pattern in BCEC-1. Asynchronous egress of both isolates was observed from 22 h post-infection onwards in BCEC-1. In addition, the tachyzoite yield (TY) was higher in F3 than in BCEC-1 infected by both isolates (P < 0.0001), highlighting better replication abilities of both parasites in F3. Nc-Spain7 showed shorter doubling times and higher TY compared to Nc-Spain1H in F3 cells; adhesion, invasion and proliferation mechanisms were very similar for both isolates in BCEC-1.
Our results indicate a highly similar behavior of high- and low-virulence isolates in their interactions with maternal caruncular cells and suggest an important role of foetal trophoblasts in the pathogenesis of N. caninum infection.
刚地弓形虫是牛流产的主要原因之一，它非常有效地穿过胎盘屏障，而胎盘损伤在流产发病机制中至关重要。牛胎盘的胎突和绒毛膜层是胎盘突中母体-胎儿界面的关键细胞成分，在胎盘功能中起着根本作用。
我们研究了高（Nc-Spain7）和低（Nc-Spain1H）毒力的刚地弓形虫分离株在牛绒毛膜上皮（BCEC-1）和滋养层（F3）细胞的建立培养物中的速殖子黏附、侵袭、增殖和逸出。通过免疫染色菌斑测定，分别在不同时间点和感染复数（MOI）下确定寄生虫入侵率（pInvR）和细胞感染率（cInfR）。此外，还通过实时 PCR（qPCR）分析和免疫染色菌斑测定，在不同时间点研究了速殖子生长动力学。
刚地弓形虫在两种细胞系中均能侵袭和增殖。Nc-Spain7 分离株在 F3 中的 pInvR 高于 BCEC-1 细胞（P<0.05），而 Nc-Spain7 分离株在 F3 细胞中的 pInvR 高于 Nc-Spain1H 分离株（P<0.01）。两种分离株在 F3 细胞中的 cInfR 也高于 BCEC-1 细胞（P<0.0001），Nc-Spain7 分离株的 cInfR 高于两种细胞系中的 Nc-Spain1H 分离株（P<0.05）。速殖子生长动力学显示，两种分离株在 F3 中均表现出速殖子指数生长，直到 58 小时达到逸出点，而 Nc-Spain1H 在 BCEC-1 中表现出非指数生长模式。在 BCEC-1 中，从感染后 22 小时开始，两种分离株均出现异步逸出。此外，与 BCEC-1 感染相比，F3 中两种分离株的速殖子产量（TY）均较高（P<0.0001），这突出了两种寄生虫在 F3 中的更好复制能力。Nc-Spain7 分离株在 F3 中的倍增时间短于 Nc-Spain1H 分离株，TY 高于 Nc-Spain1H 分离株；两种分离株在 BCEC-1 中的黏附、侵袭和增殖机制非常相似。
我们的结果表明，高毒力和低毒力分离株在与母体绒毛细胞的相互作用中表现出高度相似的行为，并表明胎牛滋养层在刚地弓形虫感染的发病机制中起着重要作用。
