This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 107 tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.

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Previous assays in pregnant animals have demonstrated the effect of different host factors and timing of infection on the outcome of neosporosis during pregnancy. However, the influence of Neospora caninum isolate itself has been poorly investigated. Here, we compared the effects on clinical outcome and vertical transmission observed in a pregnant mouse model following infection with 10 different N. caninum isolates. The isolates in our study included the Nc-Liv isolate and nine N. caninum isolates obtained from calves. Female BALB/c mice were inoculated with 2 × 106 tachyzoites at day 7 of pregnancy. Morbidity and mortality, in both dams and offspring during the course of infection, and transmission to progeny at day 30 postpartum were evaluated. The serum IgG1 and IgG2a production in dams were also examined. All dams showed elevated IgG1 and IgG2a responses, confirming N. caninum infection, although signs of disease were only exhibited in dams infected with 4 of the 10 isolates (Nc-Spain 4H, Nc-Spain 5H, Nc-Spain 7 and Nc-Liv). In neonates, clinical signs were observed in all N. caninum-infected groups, and neonatal mortality rates varied from greater than 95% with the isolates mentioned above to less than 32.5% with the other isolates. Vertical transmission rates, as assessed by parasite PCR-detection in neonate brains, also varied from 50% to 100% according to the isolate implicated. These results confirm the wide pathogenic and transmission variability of N. caninum. The intra-specific variability observed herein could help us explain the differences in the outcome of the infection in the natural host.

In this study, we examined the in vitro invasion and proliferation capacities of the Nc-Liv and ten Spanish Neospora caninum isolates (Nc-Spain 1 H - Nc-Spain 10). The invasion rate was determined as the number of tachyzoites that completed their internalisation into MARC-145 cells at 2, 4, and 6 h post-inoculation (pi). The proliferation rate was evaluated by determining the doubling time during the exponential proliferation period. Significant differences in the invasion rates of these isolates were detected at 2 and 4 h pi (P < 0.0001, Kruskal-Wallis test). At 4 h pi, the Nc-Spain 4 H and Nc-Liv isolates displayed the highest, while the Nc-Spain 3 H and Nc-Spain 1 H isolates had the lowest invasion rates (by Dunn's test). Variations in the proliferation kinetics of these isolates were also observed. Between different isolates, the lag phase, which occurs before the exponential growth phase, ranged from 8 to 44 h, and the doubling time ranged from 9.8 to 14.1 h (P = 0.0016, ANOVA test). Tachyzoite yield, which combines invasion and proliferation data, was also assessed and confirmed marked differences between the highly and less prolific isolates. Interestingly, a direct correlation between the invasion rates and tachyzoite yields, and the severity of the disease that was exhibited by infected pregnant mice in previous works could be established for the isolates in this study (Spearman's coefficient > 0.62, P < 0.05). The results of this study may help us to explain the differences in the pathogenicity that are displayed by different isolates.

Neospora caninum is considered one of the main causes of abortion in cattle, yet recent studies have also emphasised its relevance as an abortifacient in small ruminants. In order to gain deeper insight into the pathogenesis of ovine neosporosis, pregnant ewes were intravenously inoculated with 106 tachyzoites of the Nc-Spain7 isolate at days 40, 90 or 120 of gestation. Infection during the first term resulted in the death of all foetuses between days 19 and 21 post-infection, showing mainly necrotic lesions in foetal liver and the highest parasite DNA detection and burden in both placenta and foetal viscera. After infection at day 90, foetal death was also detected in all ewes, although later (34–48 days post-infection). In this group, lesions were mainly inflammatory. Foetal livers showed the lowest frequency of lesions, as well as the lowest parasite detection and burden. All ewes infected at day 120 delivered viable lambs, although 3 out of 9 showed weakness and recumbency. Neospora DNA was detected in all lambs but one, and parasite burden was similar to that observed in day 90 group. Lesions in this group showed more conspicuous infiltration of inflammatory cells and higher frequency in foetal brain and muscle when compared to both previous groups. These results highlight the crucial role that the stage of gestation plays on the course of ovine neosporosis, similar to that reported in bovine neosporosis, and open the doors to consider sheep as a valid model for exogenous transplacental transmission for ruminant neosporosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0139-y) contains supplementary material, which is available to authorized users.