Immunization with a Multivalent Listeria monocytogenes Vaccine Leads to a Strong Reduction in Vertical Transmission and Cerebral Parasite Burden in Pregnant and Non-Pregnant Mice Infected with Neospora caninum

Neospora caninumis an apicomplexan parasite that causes abortion and stillbirth in cattle.We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified versionof the attenuatedListeria monocytogenesvaccine vector Lm3Dx_NcSAG1, which expresses the majorN. caninumsurface antigen SAG1. Multivalent vaccines were generated by the insertion ofgra7and/orrop2genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Sixexperimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1×107CFU of the respective vaccine strains. Seven days post-mating, mice werechallenged by the subcutaneous injection of 1×105N. caninumNcSpain-7 tachyzoites. Non-pregnantmice, dams and their offspring were observed daily until day 25 post-partum. Immunization withLm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival,whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almostall pups had died at the end of the experiment in the infection control. The triple mutant wasthe most promising vaccine candidate, providing the highest rate of protection against verticaltransmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1resulted in lower vertical transmission and enhanced protection against cerebral infection in damsand in non-pregnant mice