Differential susceptibility of bovine caruncular and trophoblast cell lines to infection with high and low virulence isolates of Neospora caninum

Abstract

Background 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.MethodsWe 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.Results 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 (TY58h) 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 TY58h compared to Nc-Spain1H in F3 cells; adhesion, invasion and proliferation mechanisms were very similar for both isolates in BCEC-1.ConclusionsOur 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.

Crosstalk between Neospora caninum and the bovine host at the maternal-foetal interface determines the outcome of infection

“…Previously, we used in vitro and in vivo models to characterize two N. caninum isolates with marked differences in virulence: Nc-Spain7 and Nc-Spain1H, previously classified as high-and lowvirulence isolates, respectively [6][7][8]. Specifically, in bovine trophobast cells [9][10][11] and macrophages [12], Nc-Spain7 showed an increased infection and proliferation rates, whereas Nc-Spain1H displayed a reduced proliferation associated to a higher stimulation of immune responses. However, in vitro models cannot mimic the complex architecture of the bovine placenta, as they lack the microenvironmental influences and the host ability to compensate for stress conditions.…”

Integrative transcriptome and proteome analyses define marked differences between Neospora caninum isolates throughout the tachyzoite lytic cycle

“…Host tissue damage occurs as a consequence of the tachyzoite lytic cycle, a process that enables parasite propagation and involves the following successive steps: parasite invasion, adaptation to new intra-cytoplasmatic conditions, intracellular proliferation and egress from host cells [7,8]. Interestingly, the in vitro behaviour of a N. caninum population in these processes has demonstrated the potential association of the phenotypic traits such as the invasion rate and tachyzoite yield with pathogenicity observed in animal models [9][10][11]. Nevertheless, the molecular basis and mechanisms that govern such biological diversity in N. caninum remain largely unknown.…”

Proteomic Characterization of Host-Pathogen Interactions during Bovine Trophoblast Cell Line Infection by Neospora caninum

“…F3 infections for proteome analyses were performed as described previously [4]. Briefly, F3 monolayers in DMEM free of phenol red (Gibco, Gaithersburg, MD, USA) and FBS were inoculated with purified tachyzoites at an MOI of 8 for Nc-Spain7 and 10 for Nc-Spain1H in order to obtain the highest number of infected cells with a similar infection rate for both isolates [4]. Additional flasks with F3 monolayers were maintained under the same conditions but were not infected, as controls.…”

“…Recently, the bovine placental trophoblast cell line F3 has been proposed as a good in vitro model for studying the interaction of N. caninum with its target cells [4]. Using this model, we have previously shown that N. caninum infection modulates the transcriptome of host cells, especially in pathways such as extracellular matrix organization and cholesterol metabolism, favouring a pro-inflammatory response.…”