Interacciones hospedador-patógeno en la placenta durante la infección por Neospora caninum a final de la gestación: explorando los mecanismos de transmisión transplacentaria y supervivencia fetal

Esther Collantes-Fernández¹, Sandra Montaner-Da Torre¹, Montserrat Coronado-Brieva¹, Roberto Sánchez-Sánchez¹, Rafael Amieva¹, Luis Miguel Ortega-Mora¹, Pilar Hocajo¹

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

Montaner-Da Torre S^a, Coronado-Brieva M^a, Sánchez-Sánchez R^a, Amieva R^a, Ortega-Mora LM^a, Collantes-Fernández E^a, Horcajo P^a.

^a SALUVET Group, Animal Health
Department, Faculty of Veterinary Sciences, Complutense University of Madrid,
Spain.

Bovine neosporosis is a parasitic disease
caused by the protozoan Neospora caninum that represents one of the major infectious causes of abortion in cattle worldwide. The impact of transplacental infection could be foetal death or birth of a congenitally infected calf, which depends on several factors, highlighting the period of gestation at which infection occurs. As gestation progresses, there is a shift in the balance between proinflammatory and anti-inflammatory responses at the maternal-foetal interface, driven by maternal hormones. In the early stages of pregnancy, a proinflammatory environment prevails, which later transitions into an anti-inflammatory state that supports foetal growth. This dynamic immunological balance influences the disease pathogenesis and determines the pregnancy outcome. Previously, we used whole-transcriptome analysis (RNA-seq) to investigate the host-parasite interactions at the placental level following infection of heifers at mid-gestation (day 110 of gestation) with the high-virulence isolate Nc-Spain7, which induces abortion at this stage. This infection triggered severe lesions and an exacerbated proinflammatory immune response in placentomes, characterized by the overexpression of genes related to TNF-α signalling via NF-κB, IL-6/JAK/STAT3, and IL-2/STAT5 signalling, as well as IFN-α and IFN-γ responses. Additionally, the proinflammatory response in caruncles was more intense in heifers carrying dead foetuses. The combination of this inflammatory environment in the placenta and the ability of high-virulence isolates to survive and replicate appears to drive abortion at mid-gestation. However, when this high-virulence Nc-Spain7 isolate was inoculated in the last third of gestation (day 210), resulted in congenital foetal infection without causing mortality. Similar transcriptomic studies to those conducted at mid-gestation to explore host-parasite interactions during late gestation have not yet been performed. This gap raises a key question: What host molecular mechanisms and parasite factors facilitate transplacental transmission of N. caninum in
late gestation?. In this study, we aimed to investigate the mechanisms triggered
in the placenta (caruncles and cotyledons) at early stage of infection (day 20
post-infection) after challenging pregnant heifers at late-gestation with the
high-virulence isolate Nc-Spain7, using whole-transcriptome analysis.

To achieve this, pregnant Holstein-Friesian
breed heifers were inoculated at day 210 of gestation with with 10⁷ Nc-Spain7 tachyzoites (n = 5) or PBS (n = 3) and were sacrificed at day 20 post-infection. Three medial caruncles and cotyledons from each animal were separately pooled per tissue, and total RNA was extracted using the automated Maxwell® 16 System following the manufacturer’s instructions. Raw sequencing reads were aligned to the Bos taurus reference genome (ARS-UCD1.2), and
differential expression analysis between infected and non-infected animals was
performed independently for each tissue using the DESeq2 R package.

The differential expression analysis of caruncular tissue from N. caninum
infected heifers versus non-infected animals identified 46 differentially
expressed genes (DEGs) at late gestation, suggesting very subtle alterations in
this tissue. Functional enrichment analyses revealed significant
overrepresentation of pathways involved in immune response, protozoan defence,
type II interferon signalling, and T cell differentiation. Among the most
prominently upregulated genes were key chemokines (CXCL9, CXCL10),
T cell-associated markers (CD3E, CD4, CD8A),
and interferon-stimulated genes (GBP2,
GBP5), indicating a localized
immune activation in response to infection. In contrast, the foetal cotyledon
exhibited a marked transcriptomic remodelling with a total of 2,044 DEGs
identified. Enrichment analyses in this tissue highlighted the involvement of
biological processes related to angiogenesis, cellular motility and migration,
oxidative phosphorylation and phagosome.

Comparative gene set enrichment analysis (GSEA) between late and mid-gestation stages demonstrated a consistent upregulation of immune-related pathways across both gestational time points and tissues, but with a more intense response observed at mid-gestation. Moreover, distinct tissue-specific dynamics were evident. In the caruncle, the infection at mid-gestation was characterized by inhibition of epithelial–mesenchymal transition (EMT) and angiogenesis, changes that were no longer observed during the infection at late gestation. Conversely, in the foetal cotyledon, the hypoxia pathway was activated at mid-gestation but suppressed in late gestation which could contribute to the foetal survival at this stage. 

These findings contribute to a deeper understanding of the virulence and
pathogenesis of neosporosis promoting the development of effective control
measures against this disease in the future by the discovery of novel
approaches for vaccine design.

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