Neospora caninum en el último tercio de gestación: dinámica de la infección en los compartimentos materno y fetal.

Esther Collantes-Fernández¹, Sandra Montaner-Da Torre ¹, Roberto Sánchez-Sánchez¹, Rafael Amieva¹, Javier Moreno-Gonzalo², Javier Blanco-Murcia², Iván Pastor-Fernández¹, Luis Miguel Ortega-Mora¹, Pilar Horcajo¹

(1)-Saluvet, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain. (2)-Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain

Montaner-Da Torre S^a, Sánchez-Sánchez R^a, Amieva
R^a, Moreno-Gonzalo J^a,b, Blanco-Murcia J^a,b, Pastor-Fernández I ^a, Ortega-Mora LM^a, Horcajo P^a & Collantes-Fernández E^a.

^a Saluvet, Animal Health Department, Faculty of Veterinary Sciences,
Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid,
Spain.

^b Department of
Animal Medicine and Surgery, Faculty of Veterinary Sciences, Complutense
University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.

The apicomplexan parasite Neospora caninum is
considered one of the main causes of abortion in cattle worldwide and one of
the pathogens most efficiently transmitted by the transplacental route. The
outcome of infection in pregnant cattle depends on several factors,
particularly the immunological changes in both the dam and foetus at different
gestational stages. Specifically, infection during mid-gestation, when the
foetal immune system is still developing, often results in foetal death. In
contrast, infection during late-gestation, when the foetus has developed
immunocompetence, typically leads to birth of congenitally infected calves. Previously,
we used a pregnant bovine model challenged at mid-gestation with the
high-virulence Nc-Spain7 isolate to investigate early infection dynamics
associated with foetal mortality. Here, we applied the same bovine model, but
challenged at late gestation to gain a comprehensive understanding of why N.
caninum infection is less pathogenic during this period, when abortion
does not occur.

For this purpose, a total of 8 pregnant Holstein-Friesian breed heifers,
aged 2 years, seronegative for N. caninum and either free of
or vaccinated against, Leptospira spp., IBR and BVD were selected
from a herd free of brucelosis and paratuberculosis. Cattle were housed in the
experimental farm at the Faculty of Veterinary Sciences (Complutense University
of Madrid) and randomly divided into two groups: G1 (n=5)
intravenously inoculated with 10⁷ Nc-Spain7 tachyzoites on
day 210 of gestation, and G2 (n=3) inoculated with PBS (control group). After
inoculation, rectal temperatures were recorded daily from day 5 before
inoculation, until day 14 post-infection, and weekly afterwards. Foetal
viability was monitored once a week throughout the experiment by ultrasound
scanning. From day 1 post-infection until the end of the experiment, blood
samples were collected twice a week from all heifers to characterize peripheral cellular and humoral immune
responses. Serum IFN-gamma and N. caninum-specific IgG
antibodies were measured by ELISA. The percentage of monocytes, natural killer (NK) cells, B and T lymphocytes
(CD4+, CD8+ and gamma-delta) in peripheral blood mononuclear cells (PBMC) were
determined by flow cytometry. All heifers were euthanized at day 20
post-infection, and placental and foetal samples were collected for analysis.
Parasite dissemination was determined by quantitative PCR in caruncles and
cotyledons separately, and also in foetal target tissues (liver, lung and brain).
Foetal immune response was also analysed.

Infected animals exhibited fever (> 39.5° C) at some point between 3 and 6 days post-infection, with the highest mean temperature on day 3 post-infection (P < 0.05). Regarding immune response in infected
heifers, IgG seroconversion was observed as early as day 13 post-infection, with a peak of IFN-gamma production at day 6 post-infection (P < 0.05). Preliminary cytometry analysis showed in comparison to day -1, a significant increase in CD4+ T lymphocytes and NK cells on day 16 post-infection, while B lymphocytes decreased. CD4+ T lymphocytes and NK play key roles in the immune response against N. caninum, promoting both the early recognition and
elimination of pathogen-infected cells and the development of a more
specific cell-mediated response against the parasite, thereby contributing to
the initial control of the infection. The decrease in B lymphocytes
could reflect differentiation into plasma cells that secrete antibodies, which
reduces the number of B lymphocytes in dam blood. Foetal mortality
was not observed but N. caninum congenital transmission
was confirmed at day 20 post-infection by detecting parasite DNA in placental
and foetal samples from infected animals. In
fetuses, evidence of immunological competence was observed, with activation of
both humoral and cell-mediated immune responses, which may contribute to
controlling the infection. Neospora–specific IgG was detected in fluids from all fetuses (IFAT titers ranging from 1:50 to 1:200), and interferon-gamma was also detected by ELISA. Comparative analyses of infections at mid- versus late- gestation will be presented at the meeting. 

These preliminary findings highlight the influence of the gestational
period on the outcome of N. caninum infection. Notably, while
infection with a high-virulence isolate results in foetal death at
mid-gestation, it leads to congenital foetal infection without mortality at
late-gestation. Understanding the mechanisms behind both outcomes is
crucial for developing effective control strategies against N. caninum-induced
abortions.

Acknowledgements: This work was funded by the projects PID2022-139673OB-I00
(MICIU/AEI/ 10.13039/501100011033; ERDF/EU) and TEC-2024/BIO-66/SALAINDEC-CM (Community
of Madrid, Spain. Sandra Montaner-Da Torre and Rafael Amieva PRE2020-092101) were financially supported by a grant
from the Spanish Ministry of Science
and Innovation.