No abstract

Neospora caninum , a protozoan parasite closely related to Toxoplasma gondii , represents one of the main causes of abortion in cattle. Macrophages (MØs) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived MØs and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the MØs. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine MØs that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected MØs showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-γ release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in MØs infected with both isolates. Infected MØs exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected MØs, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived MØs and details isolate-dependent differences in host cell responses to the parasite.

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.

Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously inoculated with PBS (G-Control) or 107 tachyzoites of high (G-NcSpain7)- or low (G-NcSpain1H)-virulence isolates at 110 days of gestation. Serial culling at 10 and 20 days post-infection (dpi) was performed. Fever was detected at 1 dpi in both infected groups (P < 0.0001), and a second peak was detected at 3 dpi only in G-NcSpain7 (P < 0.0001). At 10 dpi, Nc-Spain7 was detected in placental samples from one animal related to focal necrosis, and Nc-Spain7 transmission was observed, although no foetal lesions were associated with this finding. The presence of Nc-Spain1H in the placenta or foetuses, as well as lesions, were not detected at 10 dpi. At 20 dpi, G-NcSpain7 animals showed almost 100% positive placental tissues and severe focal necrosis as well as 100% transmission. Remarkably, foetal mortality was detected in two G-NcSpain7 heifers. Only one animal from G-NcSpain1H presented positive placental samples. No foetal mortality was detected, and lesions and parasite transmission to the foetus were not observed in this group. Finally, 100% of G-NcSpain7 heifers at 20 dpi presented specific antibodies, while only 60% of G-NcSpain1H animals presented specific antibodies at 20 dpi. In addition, earlier seroconversion in G-Nc-Spain7 was observed. In conclusion, tachyzoites from Nc-Spain7 reached the placenta earlier and multiplied, leading to lesion development, transmission to the foetus and foetal mortality, whereas Nc-Spain1H showed delayed infection of the placenta and no lesional development or transmission during early infection.

Background Bovine neosporosis, one of the main causes of reproductive failure in cattle worldwide, poses a challenge for the immune system of pregnant cows. Changes in the Th-1/Th-2 balance in the placenta during gestation have been associated with abortion. Cotyledon and caruncle cell layers form the maternal-foetal interface in the placenta and are able to recognize and induce immune responses against Neospora caninum among other pathogens. The objective of the present work was to elucidate the immunomodulation produced by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of N. caninum in bovine trophoblast (F3) and caruncular cells (BCEC-1) at early and late points after infection. Variations in the mRNA expression levels of toll-like receptor-2 (TLR-2), Th1 and Th2 cytokines (IL-4, IL-10, IL-8, IL-6, IL-12p40, IL-17, IFN-γ, TGF-β1, TNF-α), and endothelial adhesion molecules (ICAM-1 and VCAM-1) were investigated by RT-qPCR, and protein variations in culture supernatants were investigated by ELISA. Results A similar pattern of modulation was found in both cell lines. The most upregulated cytokines in infected cells were pro-inflammatory TNF-α ( P < 0.05–0.0001) and IL-8 ( P < 0.05–0.001) whereas regulatory IL-6 ( P < 0.05–0.001) and TGF-β1 ( P < 0.05–0.001) were downregulated in both cell lines. The measurement of secreted IL-6, IL-8 and TNF-α confirmed the mRNA expression level results. Differences between isolates were found in the mRNA expression levels of TLR-2 ( P < 0.05) in both cell lines and in the mRNA expression levels ( P < 0.05) and protein secretion of TNF-α ( P < 0.05), which were higher in the trophoblast cell line (F3) infected with the low-virulence isolate Nc-Spain1H. Conclusions Neospora caninum infection is shown to favor a pro-inflammatory response in placental target cells in vitro. In addition, significant immunomodulation differences were observed between high- and low-virulence isolates, which would partially explain the differences in virulence. Electronic supplementary material The online version of this article (10.1186/s13071-019-3466-z) contains supplementary material, which is available to authorized users.