Animal procedures were approved by the Animal Welfare and Experimentation Committee of the Complutense University of Madrid and the Animal Protection Area of the Community of Madrid, Spain (PROEX 66.7/20 and 064/19), adhering to the appropriate guidelines.

The use of genetically modified organisms was approved by the Genetically Modified Organisms Committee, and its manipulation was adjusted to that described in the current legislation (Law 9/2003; Royal Decree 178/2004; Directive 2009/41/UE).

Neospora caninum tachyzoites were maintained in MARC145 cells cultures as previously described (6). Parasites were passaged onto new cell monolayers every 3 days. Tachyzoites used for in vivo and in vitro assays were recovered from flasks, when the majority of the parasites were still intracellular, and a similar passage number was used for all strains (20–25 passages). Tachyzoites used for BMDMs and for human foreskin fibroblast cells (HFF) infection were purified using PD-10 Desalting Columns (GE Healthcare, Chicago, USA), as previously described (6). The quantity and viability of tachyzoites were determined by Trypan blue exclusion followed by direct counting in a Neubauer chamber.

The highly virulent N. caninum isolate Nc-Spain7 (wild-type strain, WT) was used to generate the NcRop2 knockout (NcΔROP2) strain using the CRISPR/Cas9 system as previously described (17). Briefly, guide RNAs (gRNAs) targeting the 5’ and 3’ ends of the NcRop2 (ToxoDB ID NCLIV_001970) coding regions were designed in silico. Subsequently, gRNA sequences were introduced in the BsaI cloning site of the pSS013-Cas9 plasmid (pU6, Addgene plasmid #52694) (18). The pLoxP-mCherry-DHFR plasmid (Addgene plasmid #70147), carrying the dihydrofolate reductase–thymidylate synthase (DHFR-TS) gene that confers resistance to pyrimethamine, was used as a donor template. Both the gRNA-containing plasmids and the NotI-linearized mCherry-DHFR plasmid (5:1 insert:gRNA molar ratio) were co-transfected by electroporation into approximately 3×10⁷ tachyzoites of the Nc-Spain7 isolate. At 24 h post transfection, knockout parasites were selected in media supplemented with 10 μM pyrimethamine (Sigma-Aldrich, St. Louis, MO, USA) for a minimum of three passages. Subsequently, cloning by limiting dilution was performed and two clones selected for phenotyping in mice (see Virulence assessment in BALB/c murine model for congenital and cerebral neosporosis section).

To generate a complemented strain of NcΔROP2, the coding region of NcRop2 gene, including 1000 bp upstream and downstream of the start and stop codons, respectively, was amplified. In addition, homology arms of approximately 800 bp upstream and downstream of the CRISPR/cas cleavage site at the uracil phosphoribosyl-transferase (UPRT) gene (NCLIV_056020) were included to enhance transfection efficiency. The resulting amplicon was inserted into the multiple cloning site of the universal pUC19 plasmid, generating the pUC19-NcROP2 plasmid. Transfection was performed with a KpnI-linearized version of this plasmid and a pU6 plasmid containing a gRNA sequence targeting the 5’ end of the UPRT at a molar ratio of 1:5 (gRNA:insert). Complemented parasites were selected in media supplemented with 15 μM 5-fluorodeoxyuridine (FUDR, Sigma-Aldrich, St. Louis, MO, USA), and single clones obtained by limiting dilution.

Confirmation of the correct integration of DHFR-TS into the NcROP2 locus in the KO strains, as well as the correct deletion and insertion of the exogenous copy of NcRop2 into the UPRT locus in the complemented strains was assessed by PCR. DNA from individual clones was extracted using the Maxwell^® 16 Cell LEV DNA Purification Kit (Promega, Madison, WI, USA), and PCR reactions were performed using Taq DNA polymerase (Ecogen, Madrid, Spain) in a final volume of 25 μL, following manufacturer’s recommendations. All primers used are listed in Supplementary Table S1 . In addition, the presence or absence of NcROP2 expression was confirmed by immunofluorescence assay (see “Immunofluorescence staining” section).

The virulence of NcRop2 knockout parasites was evaluated in the well-established BALB/c model for congenital and cerebral neosporosis (19). This model is capable of detecting differences in virulence among N. caninum isolates (20). BALB/c mice (8-week-old) were procured from Janvier Labs (Laval, France) and housed in a room under a standard day/night cycle with ad libitum access to food and water. Animals were used for experimentation after 15 days of acclimatization. Induction of pregnancy in female mice was achieved through estrus-synchronization by the Whitten effect (21), and were mated for 96 h by housing one male with two females. Day 0 of pregnancy was defined as the first day females were housed with males. Subsequently, female mice were randomly distributed in 5 groups (20 females per group) and subcutaneously challenged at mid-gestation (days 7–10 of gestation) with 10⁵ tachyzoites/mouse, using two different KO clones (NcΔROP2 #8 and NcΔROP2 #42), the complemented strain (NcΔROP2::ROP2, derived from clone NcΔROP2 #42), the WT parental (Nc-Spain7) strain or PBS (unchallenged group). Mice were weighed between days 15 and 18 post-mating to confirm pregnancy, and pregnant mice were reallocated individually for delivery, whereas non-pregnant mice were housed in groups. Monitoring of clinical signs in non-pregnant mice or dams and their offspring was carried out daily until day 30 post-infection (pi) or 30 postpartum (pp), respectively. Briefly, the presence of clinical signs compatible with neosporosis were recorded using a scale with a score of 0 (no observable alterations), 1 (rough hair coat), 2 (rounded back), 3 (severe weight loss) or 4 (nervous signs), according to the description made by Pastor-Fernández et al. (15). As a humane endpoint, mice showing a body weight loss greater than 20% and nervous clinical signs were euthanized to prevent undue suffering. Non-pregnant mice were euthanized on day 30 pi in a CO₂ chamber followed by cervical dislocation, while dams and offspring were euthanized at 30 days pp. Serum and brain samples of all animals were collected and stored at −80°C for subsequent analysis.

For the N. caninum congenital model, data on fertility rate (percentage of pregnant mice), litter size (number of pups delivered per dam) and neonatal mortality (number of deceased pups from day 2 to 30 pp) were recorded throughout the experiment. The model for cerebral neosporosis was performed with the dams and non-pregnant mice at the chronic stage of infection (day 30 pp or 30 pi, respectively) by determining the parasite burden in the brain (see section “DNA extraction and qPCR parasite quantification”). Neospora caninum-specific IgG1 and IgG2 levels were determined in serum from infected mice by ELISA as described below.

We used bovine monocyte derived macrophages (BMDMs) to investigate the proliferation dynamics of N. caninum strains in a relevant bovine innate immune cell (5). BMDMs were obtained from peripheral blood drawn from a healthy adult cow as previously described (22). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated through density gradient centrifugation with Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, monocytes were isolated using mouse anti-human CD14 antibodies linked to microbeads (Miltenyi Biotec Ltd., San Diego, CA, USA), following the manufacturer’s instructions. Monocytes were seeded in 6-well plates at a density of 3×10⁶ cells/well and cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 μg/ml gentamicin, 2mM L-glutamine, 50 μM β-mercaptoethanol and 20 mM HEPES. Additionally, 100 ng/ml GM-CSF (Kingfisher Biotech Inc, St. Paul, MN, USA) was added to promote monocyte differentiation. After an incubation period of 5 days, BMDMs were recovered and re-seeded at a density of 3× 10⁶ cells/well or 3× 10⁵ cells/well in a 6-well or 24-well culture plate, respectively (22).

The proliferation kinetics of NcΔROP2 and Nc-Spain7 were determined in BMDMs by quantifying the number of tachyzoites at specific times of the lytic cycle by quantitative PCR (qPCR). Infected BMDMs were harvested and re-seeded for 24 h to acclimatize prior to infection. Subsequently, the BMDMs were inoculated with freshly syringe-lysed parasites at a multiplicity of infection (MOI) of 0.5. Non-infected MDMs were included as controls. At different time points during the lytic cycle of N. caninum (8, 24, 48, 60 and 72 hours pi), BMDMs were collected by adding 180 μl of lysis buffer (Qiagen, Germany) and 20 μl of proteinase K (Qiagen, Germany) to each well, transferred into DNAse free 1.5 ml tubes, and frozen at −80°C until used for DNA extraction for parasite quantification. All analyses were performed in 6 biological replicates obtained from 2 independent experiments, each separated by a minimum of 2 weeks.

In parallel, replicates of BMDM cultures grown on coverslips were identically infected and fixed at the same time points. Fixed cells were labelled by single immunostaining (as described below) to microscopically study the proliferation kinetics. In addition, vacuole sizes and cell infection rates (cIR: percentage of cells infected with at least one tachyzoite) were determined at 48 hours pi, when tachyzoites are still largely intracellular by immunofluorescence (see “Immunofluorescence staining” section). At least five fields were observed per coverslip.

To determine the parasite burden in the brain of infected dams, DNA from brain tissue samples (50 to 100 mg) was extracted using the Maxwell^® 16 Mouse Tail DNA Purification Kit (Promega Madison, WI, USA) and DNA concentration was determined by spectrophotometry using a nanophotometer (NanoPhotometer^®, Implen GmbH, Munich, Germany). DNA was extracted from BMDMs using the DNeasy Blood & Tissue Kit (Qiagen, Germany) following manufacturer’s instructions.

For all samples, parasite quantification was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The Nc5 region was used to quantify parasite DNA and the 28S rRNA gene was used to quantify host DNA in mice (23). Parasite burden was determined using a standard curve of 10^–1 to 10⁵ tachyzoites, and subsequently normalization to the host DNA. All primers used are listed on Supplementary Table S1 .

To evaluate the presence of NcROP2 in the KO and complemented strains, immunofluorescence test (IFAT) was performed following the protocol by Pastor-Fernández et al. (13) with minor modifications. Infected MARC-145 cells were washed three times with PBS and fixed using ice-cold methanol for ten minutes. Subsequently, cells were blocked and permeabilized in PBS with 3% BSA and 0.25% Triton-X 100 for 45 minutes at 37°C. Cultures were then incubated with the monoclonal antibody α-NcSAG1 (1:250) (24) as a surface marker and polyclonal antibody α-ROP2 (1:100) (13) for 1 h at 37°C. As secondary antibodies, Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Life Technologies, Carlsbad, CA, USA) at a dilution of 1:1000 were used for 1 h at 37°C. Nuclei were stained with DAPI (1:10.000). For infected BMDMs, a double immunofluorescence staining was performed. Cultures were initially fixed with 0.05% glutaraldehyde and 3% paraformaldehyde. After permeabilization with Triton X-100, cells were incubated with N. caninum-infected mouse serum (1:2000) as a primary antibody (25) for 1 h at 37°C, followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Life Technologies, Carlsbad, CA, USA) (1:750) for 1 h at 37°C. For macrophage staining, Alexa Fluor-594 Phalloidin (Life Technologies, Carlsbad, CA, USA) was used for 30 min at 37°C. Nuclei were stained with DAPI at 1:10000 in PBS. Images for each condition were obtained using an inverted fluorescence microscope (Nikon Eclipse TE200) equipped with a PCO.panda scientific CMOS camera. Imagining was performed at 40 ✕ or 100 ✕ magnification, and data processed through the Imaging Software NIS Elements (v. 5.30.04). Only linear adjustments to brightness and contrast were applied uniformly across each image, without altering the integrity or interpretation of the data.

N. caninum specific IgG1 and IgG2 serum levels were determined in female mice using ELISA as previously described (19). N. caninum soluble tachyzoite antigen was coated onto 96-well plates (0.125 μg/well), and ELISA was performed using a 1:100 dilution of serum samples. Peroxidase-conjugated anti-mouse IgG1 or IgG2a were used as secondary antibodies (1:5000; Southern Biotechnology, Birmingham, AL, USA). As controls, sera from mice experimentally infected with Nc-Spain7 and non-infected mice from previous experiments were used (5). Absorbance was measured at 405 nm for each plate, and results were expressed as a relative index percentage (RIPC) following the formula: RIPC = (OD sample – OD negative control)/(OD positive control – OD negative control) ✕ 100.

Differences in mortality rates were evaluated using Fisher’s F test, while the Kaplan-Meier survival method was used to estimate the percentage of surviving animals at each time point. The log-rank (Mantel-Cox) test was used to compare survival curves and calculate the median survival time. Clinical sign scores and parasite burden between groups were analyzed using the Kruskal-Wallis test followed by Dunn’s multiple-comparison test. Litter size and antibody levels were compared using one-way ANOVA followed by Tukey’s multiple-comparison test, preceded by D’Agostino-Pearson test for normal distribution. Regarding cIR, parasitic vacuole size and proliferation data, a parametric one-way ANOVA test followed by Dunnett’s test for group comparisons was used. For mRNA levels, a student t test was performed to compare groups. Statistical significance was defined at p < 0.05 for all analyses. All statistical analysis were conducted using the GraphPad Prism v.7.0 software (San Diego, CA, USA).

To unravel the functional role of NcROP2, we efficiently generated two different NcΔROP2 strain clones. The NcΔROP2 #42 clone had most of the NcRop2 coding region replaced by the DHFR-TS cassette ( Figure 1A ), while clone NcΔROP2 #8 presented a 7 bp deletion at the 5’ end of the gene, generating a frameshift mutation. To validate the successful gene deletion, we performed PCR analyses using primers targeting the NcRop2 flanking regions, as well as the DHFR-TS cassette ( Supplementary Table S1 ). Our results showed the presence of amplified bands in the parasites with disruptions in NcΔROP2 while being absent in the parental strain ( Figure 1B ). The gene disruption was also confirmed by Sanger sequencing and the absence of NcROP2 protein expression was evaluated by IFAT ( Figure 1B ).

To generate a complemented strain, we used the NcΔROP2 #42 clone as the parental strain, as it had most of the NcRop2 gene removed. We constructed complemented strains by reintroducing an exogenous copy of the target gene NcRop2 into the UPRT locus via double homologous recombination, using overhangs in the NcROP2 repair template homologous to the 5’ and 3’ UTR region of the UPRT locus to enhance efficiency. Single clones of the NcRop2 complemented strain were obtained by limiting dilution, and the expression of NcROP2 protein assessed by IFAT, with expression levels mirroring those observed in the Nc-Spain7 strain ( Figure 1B ).

Collectively, these data confirm the successful generation of NcΔROP2 KO strains, as well as their corresponding complemented strain (NcΔROP2::ROP2).

In the N. caninum congenital murine model, pups born from dams infected with either of the NcΔROP2 clones exhibited a significant increase in median survival times (19 and 20 days respectively) in comparison to the Nc-Spain7-infected group (16 days) (ρ < 0.01, log-rank test; Figure 2A ) ( Table 1 ). NcΔROP2-infected groups showed significantly lower mortality rate, with 33% and 28% of the pups surviving at the end of the experiment (day 30 pp) for clones #42 and #8, respectively, while all other infected groups reached postnatal mortality rates close to 100% ( Table 1 ). Furthermore, the complementation of NcΔROP2 led to a restoration of parasite virulence, as the offspring from the NcΔROP2::ROP2 group showed no significant differences in median survival time nor mortality rate compared to those from the Nc-Spain7 group (ρ > 0.05, log-rank test). Statistical analysis revealed no significant differences in rates of pregnancy (ρ > 0.05, Fisher’s F test) or litter size (ρ > 0.05, one-way ANOVA) among mice infected with the different strains ( Table 1 ).

In the dams, clinical signs of neosporosis became evident starting from two weeks post infection. Initial manifestations included rough hair coat and apathy, followed by anorexia, inactivity, and eventually, neurological signs. Notably, groups infected with either of the NcΔROP2 clones exhibited significantly milder clinical signs (ρ < 0.05, Kruskal-Wallis, Dunn’s comparison post-test; Figure 2B ). Furthermore, as detailed in Table 1 , lower parasite burdens were observed in dams infected with the NcΔROP2 clones compared to those infected with the Nc-Spain7 wild type strain ( Figure 2C ). Similarly, in non-pregnant mice, a significant lower parasite burden was observed in those infected with the NcΔROP2 clones compared to the Nc-Spain7 group (ρ < 0.01. Data not shown).

Significantly higher levels of specific anti-N. caninum IgG1 and IgG2a antibodies were detected in all infected groups compared to the negative control group (ρ < 0.0001, one-way ANOVA Tukey’s comparison post-test), confirming N. caninum infection (data not shown). Dams infected with the NcΔROP2 clones exhibited a significantly lower IgG1/IgG2a ratio (ρ < 0.05; one-way ANOVA Tukey’s comparison post-test), while complementation of NcRop2 restored the ratio to levels comparable to Nc-Spain7 ( Figure 2D ).

Investigations of the consequences of the NcROP2 deletion on the parasite lytic cycle in BMDMs were carried out using only the NcΔROP2 #42clone, since both #8 and #42 exhibited a similar phenotype in in vivo experiments, and #42 was the parental strain for complementation. Additionally, the NcΔROP2::ROP2 complemented strain was not tested because it displayed a phenotype similar to the WT Nc-Spain7 strain, further confirming that the observed differences were specifically due to NcRop2 disruption.

The KO strain (NcΔROP2 #42) initially displayed a proliferation pattern similar to that of the Nc-Spain7 strain, characterized by an exponential growth up to 48 hours pi. However, a notable divergence became evident beyond this point, when the NcΔROP2 strain exhibited a significantly slower growth at 60 hours pi compared to the Nc-Spain7 parasites (ρ < 0.001) ( Figure 3A ).

Microscopic examination revealed that both the WT and KO strains successfully infected host cells by 8 hours pi, as intracellular parasites were already visible within BMDMs. By 24 hours pi, multiplication had begun, and parasitophorous vacuoles started forming, reaching their maximum size between 48 and 60 hours pi ( Figure 3B ). At this stage, no significant differences in the size of parasitophorous or percentage of infected macrophages were observed (data not shown). By 72 hours pi, host cells ruptured and tachyzoites were released in the Nc-Spain7 strain. In contrast, the NcΔROP2 strain exhibited a slightly delayed egress, with a portion of parasites remaining intracellular longer than their WT counterparts, as can be seen by the presence of parasite vacuoles ( Figure 3C ). Additionally, an increased presence of vacuoles containing partially degraded tachyzoites or multinucleated complexes were observed ( Figure 3B ), suggesting that either the KO parasites were more prone to host-mediated degradation or that the egress process was incomplete. At 72 hours pi, some macrophages retained remnants of parasitic structures characterized by irregular morphology, loss of defined boundaries, and fragmented or diffuse staining patterns. These features are consistent with intracellular degradation processes previously described in N. caninum (39) and reinforce the idea that NcΔROP2 mutants face challenges in maintaining their intracellular cycle and exiting efficiently.

Our results demonstrated that in the absence of NcROP2, parasites have attenuated virulence in mice and reduced growth in BMDM compared to the parental strain Nc-Spain7. To investigate the mechanisms underlying these phenotypic traits, we performed a comparative transcriptomic analysis of BMDMs infected with NcΔROP2 and Nc-Spain7 strains. The RNA-seq analysis revealed significant transcriptional differences at both the host and parasite levels. Principal Component Analysis (PCA) of host expression data ( Figure 4A ) demonstrated distinct clustering between infected and non-infected samples, underscoring a strong transcriptional response to infection. Genes such as STAR, RASGEF1A and TMIGD3 were significantly downregulated in both comparisons ( Figure 4B ), pointing to the suppression of immune response and intracellular signaling related pathways. On the other hand, genes such as ACSS2, ACAT2, ASNS and HMGCS1, which are involved in lipid metabolism and cholesterol biosynthesis, were significantly upregulated, indicating a metabolic shift in infected cells.

In host transcriptional profiles ( Figure 4C ), NcΔROP2-infected macrophages exhibited 528 upregulated and 515 downregulated genes compared to non-infected cells ( Supplementary Table S2 ). Of these, 92 upregulated and 84 downregulated genes were unique to NcΔROP2 relative to non-infected cells. This unique set of DEGs suggests that these changes are due to the absence of ROP2 rather than the general effects of N. caninum infection. Interestingly, one of the unique DEGs for NcΔROP2 infected cells is GBP1, which encodes a key interferon-stimulated gene (ISG) involved in host defense against intracellular pathogens. Despite these differences, the Pearson correlation coefficient (R = 0.85, p < 2.2e−16) between the log2FC values of Nc-Spain7- and NcΔROP2-infected macrophages indicates a strong overall similarity in host response patterns ( Figure 4D ).

To further explore these transcriptional changes, we conducted a functional enrichment analysis of DEGs ( Supplementary Figure S1 ). Upregulated genes were primarily associated with pathways related to cell division, DNA replication and glycolysis, suggesting that both strains reprogram host metabolism to support parasite replication. Downregulated genes were predominantly related to immune processes, such as T cell activation, cytokine production and leukocyte differentiation, highlighting a shared strategy of immune evasion.

Next, Gene Set Enrichment Analysis (GSEA) was performed using the curated Hallmark gene set database ( Figure 4E ), to gain further insight into these transcriptional differences. NcΔROP2-infected BMDMs exhibited enrichment of gene sets related to metabolic pathways, including MYC targets, oxidative phosphorylation, and fatty acid metabolism. Conversely, BMDM infected with Nc-Spain7 showed enrichment in gene sets associated with cell cycle processes, such as E2F targets, G2/M checkpoint, and mitotic spindle, suggesting a focus on host cell cycle modulation.

Downregulated gene sets also revealed suppression of immune-related pathways in both strains, including inflammatory response, IL2 STAT5 signaling and TNFA signaling, underscoring their ability to inhibit pro-inflammatory and immune signaling pathways. Interestingly, interferon-related pathways, such as interferon-alpha (IFN-α) response and interferon-gamma (IFN-γ) response, were more strongly upregulated in NcΔROP2-infected BMDM, suggesting that while general immune activation was suppressed, interferon signaling pathways were more activated in the absence of ROP2.

To test the hypothesis that NcΔROP2-infected cells ineffectively suppress interferon signaling, we focused on host genes associated with IFN-γ response that change in response to NcΔROP2 infection ( Figure 4F ). All DEGs exclusively upregulated in BMDM infected with NcΔROP2 included ISGs such as CASP1, MX1, PNPT1 and IFI44. These results support the notion that the deletion of NcΔROP2 impairs the parasite ability to disrupt interferon signaling, a critical component of the host innate immune response.

To identify potential regulators driving the transcriptional changes observed in infected macrophages, DecoupleR was used to perform transcription factor (TF) enrichment analysis ( Figure 4G ). In NcΔROP2-infected BMDM, reduced activity was observed for immune-related TFs including SPI1 and KLF13, consistent with the downregulation of immune-related genes. By contrast, Nc-Spain7-infected BMDM displayed higher enrichment for TFs such as SREBF1/2, which regulate lipid metabolism, and the E2F family, key regulators of cell cycle progression. These findings align with the distinct transcriptional profiles induced by each strain.

Analysis of parasite-specific transcripts also revealed significant differences between the KO and WT strains. PCA plots of parasite gene expression revealed clear separation between Nc-Spain7 and NcΔROP2, highlighting their transcriptional divergence ( Figure 5A ). In NcΔROP2, 84 genes were upregulated, while 63 genes were downregulated in Nc-Spain7 ( Figure 5B ) ( Supplementary Table S3 ). Among the upregulated genes in NcΔROP2 ( Figure 5C ) were histones such as H2A and H2B, suggesting changes in processes like chromatin remodeling and stress response. Additionally, bradyzoite-related genes, including RON2L1 and SRS44, were upregulated in NcΔROP2, suggesting a transcriptional shift toward a latent or stress-adapted stage, likely reflecting the parasite efforts to adapt to the absence of ROP2.

By contrast, NcΔROP2 parasites displayed downregulation of key genes involved in host cell interaction, including rhoptry proteins (e.g., RDF2) and surface antigens (e.g., SAG3 and SRS34A). These molecules are critical for host cell manipulation and invasion, and their reduced expression highlights the functional consequences of ROP2 deletion. Collectively, these results illustrate the distinct transcriptional consequences in the absence of NcROP2 and how the parasite adapts to this genetic landscape.

To validate the transcriptomic results, we performed two independent assays: one to confirm the host transcriptomic findings and another to validate the parasite-related results. Since our transcriptomic analysis revealed differential activation of IFN-related pathways in BMDMs infected with NcΔROP2, we assessed their susceptibility to IFN-γ at both 48 hours pi and 60 hours pi ( Figure 6A ). At 48 hours pi, both Nc-Spain7 and NcΔROP2 exhibited a similar dose-dependent reduction in parasite growth, with significant inhibition observed at 10 ng/ml of IFN-γ (ρ < 0.01, one-way ANOVA test, Dunnett’s comparison post-test). However, at 60 hours pi, while Nc-Spain7 continued to show a clear dose-dependent inhibition, NcΔROP2 displayed a marked resistance to IFN-γ, with only minimal inhibition at the highest concentration (around 30%). Additionally, direct comparisons between Nc-Spain7 and NcΔROP2 at the same IFN-γ doses revealed significant differences at 60 hours pi (ρ < 0.01, one-way ANOVA test, Dunnett’s comparison post-test), highlighting the impact of NcRop2 deletion on parasite susceptibility to IFN-γ.

To further validate the transcriptomic findings that suggested an overexpression of bradyzoite-related genes in the NcΔROP2 KO strain, we performed a tachyzoite-to-bradyzoite conversion assay and measured the NcSag4 bradyzoite-specific gene expression at different time points following SNP stimulation (1, 3, 5, and 7 days) ( Figure 6B ). Over the course of the assay, NcSag4 transcript levels progressively increased in both groups. However, significant differences were observed between the two groups at five and seven days post-infection, with a higher fold change in the NcΔROP2 parasite (ρ < 0.01-0.05; Fisher’s F test). By contrast, NcSag1 mRNA expression remained stable throughout the assay, with no significant differences between the initial and final time points for either of the strains (data not shown).