Comparison of host cell invasion and proliferation among Neospora caninum isolates obtained from oocysts and from clinical cases of naturally infected dogs

• Tachyzoite intracellular proliferation is associated with N. caninum pathogenesis.
• Tachyzoite proliferation capacities have been related to bovine isolate virulence.
• Invasion and tachyzoite yield in vitro were evaluated in canine isolates.
• Differences were found between isolates from oocysts and from dogs with signs.
• Correlation was found between proliferation capacities and virulence in mice.

In a previous study we have shown that the in vitro invasion rate (IR) and tachyzoite yield (TY) are associated with the virulence phenotypes of Neospora caninum isolates of bovine origin. In addition, we recently observed marked differences in virulence when canine isolates were compared in a pregnant BALB/c mouse model. In this study, we investigated whether invasion and proliferation capacities could be used as virulence-related N. caninum phenotypic traits. Of the isolates compared in mice, four canine isolates obtained from oocysts (Nc-Ger2, Nc-Ger3, Nc-Ger-6, Nc-6 Arg) had shown a low-moderate virulence, and two further isolates obtained from dogs with neurological signs (Nc-Bahia, Nc-Liv) were highly virulent. The IR for each isolate was determined by a plaque assay and the counting of immunofluorescence-labeled parasitophorous vacuoles at 3 days post-inoculation (p.i.). The TY was determined by the quantification of tachyzoites at 56 h p.i. by real-time PCR. Most of the canine isolates showed similar IR values under controlled invasion conditions for 4 h and 72 h p.i., indicating a limited time period for invasion similar to that observed for bovine isolates. The Nc-Ger3, Nc-Bahia, and Nc-Liv isolates showed a significantly higher IR and TY than the Nc-Ger2 and Nc-Ger6 isolates (P < 0.0001). A correlation was found between the IRs and TY (ρ > 0.885, P < 0.033), as well as between the TY and both dam morbidity (ρ = 0.8452, P < 0.033) and pup mortality (ρ > 0.8117, P < 0.058) in mice. These results demonstrate the importance both the invasive and proliferative capacities have on the virulence of canine N. caninum isolates.

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