Host–parasite interface in Neospora caninum: Intracellular survival, immune restriction, and pathogenesis

Introduction

Neospora caninum is an obligate intracellular apicomplexan parasite causing neosporosis in a broad range of domestic and wild animals (Dubey, 2003; Donahoe et al., 2015) [2,1] . The disease was first recognized as cyst-forming protozoal encephalomyelitis and myositis in dogs and was later distinguished from toxoplasmosis with recognition of N. caninum as a separate parasite (Bjerkås et al., 1984; Dubey et al., 1988) [3,4] . Although many mammals can be infected, cattle and dogs are of greatest recognized veterinary importance: bovine neosporosis is dominated by reproductive losses, whereas canine disease commonly involves neuromuscular and neurological manifestations (Dubey et al., 2007; Fisher et al., 2024) [5,6] . Detection of Neospora-associated infection in aborted bovine fetuses established its importance as a cause of reproductive disease (Anderson et al., 1991) [7] , and congenitally infected cattle have an increased risk of abortion, including in subsequent pregnancies (Thurmond et al., 1997) [8] . Recent systematic evidence continues to show frequent N. caninum detection among aborting cattle and aborted fetuses worldwide (Nayeri et al., 2022) [9] . Consequently, abortion, fetal loss, reduced reproductive performance, animal replacement, and control measures contribute to the economic importance of bovine neosporosis (Trees et al., 1999; Hall et al., 2005) [11,10] . N. caninum has a heteroxenous life cycle involving canids as definitive hosts and numerous mammals as intermediate hosts (Dubey et al., 2007; Goodswen et al., 2013) [5,12] . Domestic dogs shed oocysts after consuming infected tissues, while coyotes and dingoes are also definitive hosts, indicating contributions from both domestic and sylvatic canids (McAllister et al., 1998; Gondim et al., 2004; King et al., 2010) [14,13,15] . Associations between infection in farm dogs and cattle support the importance of the cattle-canid interface, and dogs can shed oocysts after ingesting naturally infected bovine placental tissue (Wouda et al., 1999; Dijkstra et al., 2001) [17,16] . Intermediate hosts acquire infection after ingesting sporulated oocysts;

rapidly proliferating tachyzoites then disseminate and may differentiate into slowly replicating bradyzoites within persistent tissue cysts (Dubey et al., 2007; Goodswen et al., 2013) [12] . In cattle, transplacental transmission is particularly important and can efficiently maintain infection across generations (Davison et al., 1999) [18] . Transmission may result from recrudescence of persistent maternal infection during pregnancy, termed endogenous transplacental transmission, or from new infection acquired during gestation, termed exogenous transplacental transmission (Trees et al., 2005; Williams et al., 2009) [19,20] . Persistent maternal infection therefore maintains the parasite within cattle lineages, whereas environmental oocyst exposure can introduce new infections (Davison et al., 1999; Williams et al., 2009) [18,20] . The ability of N. caninum to persist, disseminate, and cause disease depends closely on parasite-host interactions (Dubey et al., 2007; Goodswen et al., 2013) [5,12] . Successful infection requires host-cell invasion, intracellular replication, dissemination, and persistence, while host immune responses simultaneously restrict parasite multiplication and spread (Goodswen et al., 2013) [12] . This balance is especially important during pregnancy because fetal infection does not invariably cause abortion; outcome reflects interactions among parasite transmission, persistence and replication, maternal and fetal responses, and gestational timing (Hall et al., 2005; Williams et al., 2009) [10,20] . Understanding these interactions is therefore essential for explaining intracellular survival, immune evasion or resistance, persistence, neurological disease, placental infection, fetal injury, and congenital transmission (Dubey et al., 2007; Goodswen et al., 2013) [12] . Accordingly, this review examines host-cell invasion and intracellular survival, host immune responses and parasite immune-evasion strategies, and progression from host-parasite interaction to neosporosis pathogenesis, with emphasis on molecular determinants of parasite survival and virulence, host restriction mechanisms, vertical transmission, disease severity, and potential host-and parasite-directed interventions.

Host-cell invasion and intracellular survival of Neospora caninum

Host-cell invasion by N. caninum begins with attachment of extracellular tachyzoites to the host-cell surface and coordinated secretion of apical invasion proteins. NcMIC 1 binds sulfated host-cell glycosaminoglycans, whereas NcMIC 3 is secreted onto the tachyzoite surface and promotes adhesion through its EGF-like domains (Keller et al., 2002; Naguleswaran et al., 2001) [21,22] . Other microneme proteins further support this process. NcMIC 2 is a TRAP-family protein with adhesive domains and undergoes temperature-and Ca 2+ regulated secretion, consistent with a proposed role in attachment and invasion (Lovett et al., 2000) [23] . More direct functional evidence comes from NcMIC 8 , whose interaction with NcMIC 3 and antibodymediated inhibition reduces invasion, and NcAMA 1 , an apically localized protein against which specific antibodies inhibit host-cell entry (Wang et al., 2017; Zhang et al., 2007) [24,25] . Genetic disruption of NcMIC 6 similarly decreases invasion and egress and reduces expression and secretion of NcMIC 1 and NcMIC 4 , indicating functional cooperation among components of the microneme system (Wang et al., 2021) [26] . Once entry is initiated, rhoptry proteins extend parasite manipulation beyond adhesion: NcROP 16 promotes host STAT3 phosphorylation, whereas NcROP 5 deletion reduces invasion and intracellular growth, showing that distinct rhoptry effectors contribute to early host manipulation and parasite fitness (Ma et al., 2017; Ma et al., 2017) [27,28] . These sequential events, from initial host-cell attachment and microneme/rhoptry secretion to active invasion and formation of the parasitophorous vacuole, are summarized in Figure 1. ) [29] . Several GRAs appear to support post-invasion development rather than initial entry. Disruption of NcGRA 6 reduces intracel lular replication without markedly affecting invasion or egress, while NcGRA 2 colocalizes with NcGRA 6 within the intravacuolar network (IVN) and its deletion similarly slows intracellular growth (Zhao et al., 2020; Dong et al., 2021a) [31,30] . NcGRA 12 is also present in soluble and membrane-associated forms within the PV and localizes to IVN membranes, further illustrating the structural complexity of the vacuolar niche, although its specific contribution to parasite survival remains unresolved (Dong et al., 2021b) [32] . Dense-granule proteins can additionally influence host responses: NcGRA 7 modulates host signaling and cytokine expression, and its deletion reduces parasite burden and virulence in mice without substantially altering initial infection or intracellular growth in vitro (Nishikawa et al., 2018) [33] . Thus, microneme, rhoptry, and dense-granule proteins function sequentially and in complementary ways, linking adhesion and invasion to host manipulation and organization of the intracellular replicative niche. The PV is not an isolated compartment but interacts extensively with host-cell organelles and trafficking pathways. N. caninum reorganizes host microtubules and recruits the endoplasmic reticulum, mitochondria, Golgi-associated structures, multivesicular bodies, and endolysosomal compartments around the PV (Nolan et al., 2015) [34] . These interactions are closely linked to nutrient acquisition: the parasite depends on host-derived cholesterol, accesses cholesterol associated with NPC1-containing endocytic organelles, and intercepts Rab14positive Golgi vesicles carrying sphingolipids (Nolan et al., 2015) [34] . Thus, N. caninum maintains selective communication with host trafficking systems to obtain metabolites required for growth while remaining within a protected intracellular compartment. Localization of NcGRA 2 , NcGRA 6 , and NcGRA 12 within the IVN provides a structural context for this parasite-host interface, although direct roles of individual GRAs in specific nutrient-acquisition pathways remain to be established (Dong et al., 2021a, 2021b; Zhao et al., 2020) [30,32] . Successful intracellular replication is also associated with modification of host metabolism, signaling, apoptosis, and autophagy. Infection enhances host glycolysis through a JNK-HIF-1 α -PFKFB3associated pathway, and inhibition or knockdown of PFKFB3 reduces parasite propagation, demonstrating that host metabolic reprogramming can favor intracellular growth (Tao et al., 2025) [35] . Secreted effectors provide additional routes for host manipulation: NcROP 16 activates STAT3, whereas NcGRA 7 alters signaling and cytokine responses (Ma et al., 2017; Nishikawa et al., 2018) [27,33] . Effects on apoptosis appear context dependent. Infection can inhibit death-receptor-mediated apoptosis and caspase-3 activation, potentially prolonging host-cell viability, whereas NcROP 16 has been associated with increased expression of apoptosis-related factors under different experimental conditions (Herman et al., 2007; Ma et al., 2017) [36,27] . Autophagy shows similar context dependence. In macrophage-based models, infection is associated with accumulation of autophagic structures, impaired autophagic flux, and inefficient autophagosome-lysosome fusion through TLR2-and mTOR-associated signaling (Wang et al., 2021) [37] . In contrast, infection of caprine endometrial epithelial cells enhances autophagic flux through mTOR inhibition, and experimentally promoting autophagy increases tachyzoite propagation (Zhao et al., 2022) [38] . N. caninum also induces mitophagy in macrophages; enhanced mitophagy reduces ROS and proinflammatory responses and is associated with increased parasite burdens, suggesting another mechanism by which host homeostatic pathways can be redirected in favor of parasite persistence (Zhang et al., 2022) [39] .

Maintenance of this intracellular niche ultimately depends on preserving a vacuolar environment that limits destructive lysosomal activity while retaining selective interactions with host organelles. The N. caninum PV is generally considered largely non-fusogenic and resistant to conventional lysosomal degradation (Yang et al., 2021) [29] , although lysosomal and endosomal structures can accumulate around the vacuole as part of broader host-organelle rearrangement (Nolan et al., 2015) [34] . This distinction suggests that intracellular survival depends not on complete exclusion of the endolysosomal system but on preventing productive fusion and acidification that would destroy the parasite. Supporting this interpretation, upregulation of GIMAP4, GIMAP5, or GIMAP6 promotes LAMP1 accumulation at the PVM, PV acidification, and reduced parasite proliferation in rat and bovine cells (Azam et al., 2025) [40] . Together with the reduced post-invasion growth observed after disruption of NcROP 5 , NcGRA 2 , or NcGRA 6 (Ma et [28,30,31] , these findings indicate that N. caninum intracellular survival is multifactorial, integrating formation of a specialized PV, secretion of parasite effectors, exploitation of host nutrients, modulation of metabolic and degradative pathways, and protection of the vacuolar niche from destructive lysosomal activity.

Host immune responses and parasite immune-evasion strategies

Recognition of N. caninum by the innate immune system initiates a predominantly type 1 inflammatory response involving macrophages, dendritic cells, and other innate effector populations. TLR signaling contributes substantially to this recognition: TLR2 promotes antigenpresenting-cell activation and subsequent Th1 polarization, whereas TLR3 recognizes parasite-derived RNA through TRIF-dependent signaling that promotes IL-12, TNF-α, type I interferons, and other inflammatory mediators (Mineo et al., 2010; Miranda et al., 2019) [41,42] . Consistently, MyD88-deficient mice show impaired dendritic-cell IL-12 production, delayed IFN-γ responses, and increased susceptibility, linking innate sensing directly to protective cytokine responses (Mineo et al., 2009) [43] . Intracellular recognition also involves NOD2, whose deficiency reduces IL-6 and TNF-α production and impairs parasite control, although NOD2-driven inflammation may additionally contribute to immunopathology (Davoli-Ferreira et al., 2016) [44] . In murine macrophages, TLR11-dependent MEK-ERK signaling promotes IL-12 p40 production, providing another connection between recognition and type 1 immunity, although this pathway should be interpreted specifically within the murine model (Jin et al., 2017) [45] . Both conventional and plasmacytoid dendritic cells produce IL-12 early after infection, linking parasite sensing with IFN-γ-mediated immunity (Teixeira et al., 2010) [46] . Macrophages likewise have a dual role as parasite host cells and immune effectors: tachyzoites can invade these cells, yet macrophage recruitment and activation contribute substantially to protection, as their depletion markedly increases susceptibility to infection (Dion et al., 2011; Abe et al., 2014) [48,47] . In cattle, NK cells provide an additional early defense by producing IFN-γ in response to N. caninum and killing infected fibroblasts predominantly through perforin-dependent mechanisms, although NK cells themselves can also be invaded by the parasite (Boysen et al., 2006) [49] . Collectively, these findings indicate that multiple recognition pathways converge on antigen-presenting-cell activation, IL-12 production, and early IFN-γ responses. The major innate, adaptive, and cell-autonomous host immune mechanisms involved in the control of N. caninum infection are summarized in Table 1. . This type 1 response is also evident in cattle, where experimental infection induces rapid parasite-specific lymphocyte proliferation accompanied by strong IFN-γ production (Lundén et al., 1998) [51] . Downstream of IFN-γ, macrophages restrict intracellular replication through inducible nitric oxide synthase and nitric oxide, since interference with this pathway diminishes parasite control (Tanaka et al., 2000b) [52] . IFN-γ also induces indoleamine 2,3-dioxygenase in bovine and human cells, causing tryptophan depletion and inhibition of parasite growth; reversal by tryptophan supplementation or IDO inhibition confirms nutrient deprivation as another cell-autonomous mechanism (Spekker et al., 2009) [53] . Oxidative and inflammasomedependent defenses provide an additional layer, as N. caninum activates inflammasome components together with ROS-associated responses, and disruption of these pathways compromises restriction of intracellular replication (Mota et al., 2020) [54] . Adaptive immunity reinforces these mechanisms. CD4 + T-cell depletion increases mortality and reduces parasite-specific antibody responses, whereas high antibody titers cannot compensate for loss of IFN-γ during acute infection, indicating that humoral immunity functions most effectively alongside cellular responses (Tanaka et al., 2000a) [55] . CD8 + T cells also contribute to protection: CD8-deficient mice carry higher parasite burdens, and adoptive-transfer experiments indicate that their protective activity depends predominantly on IFN-γ rather than perforin-mediated cytotoxicity (Correia et al., 2015) [56] . TLR2-dependent innate activation influences both CD4 + and CD8 + responses by supporting parasite-specific proliferation and an appropriate IFN-γ/IL-10 balance (Mineo et al., 2010) [41] . In murine cells, IFN-γ additionally induces immunity-related GTPases such as Irga 6 that accumulate on the N. caninum PVM; unlike virulent Toxoplasma gondii, N. caninum lacks a fully functional ROP18-mediated mechanism for neutralizing this response, illustrating direct IFN-γ-dependent targeting of the parasite vacuole in this model (Reid et al., 2012) [57] . Thus, IFN-γ integrates systemic immunity with nitric oxide production, nutrient restriction, oxidative responses, T-cell effector functions, and, in mice, PVM-directed intracellular defense. Despite these protective responses, N. caninum can modify host signaling and homeostatic pathways in ways that favor persistence. Parasite-induced p38 MAPK activation in macrophages appears to dampen protective activation, because p38 inhibition increases IL-12 p40 production and MHC/B7 expression and, in vivo, is associated with stronger inflammatory responses, reduced parasite burden, and improved survival (Mota et al., 2016) [58] . Infection-induced mitophagy provides another mechanism: enhanced mitochondrial removal reduces ROS and suppresses ERK-, p38-, and NLRP3-associated inflammatory responses, whereas experimental promotion of mitophagy increases parasite burden (Zhang et al., 2022) [39] . This contrasts with the protective ROS-inflammasome interaction observed during infection and illustrates how parasite manipulation of host homeostatic pathways can shift the balance away from intracellular restriction (Mota et al., 2020; Zhang et al., 2022) [54,39] . Host cells nevertheless possess mechanisms capable of directly overcoming the protected PV. Upregulation of GIMAP4, GIMAP5, or GIMAP6 promotes LAMP1 accumulation at the PVM, vacuolar acidification, and restriction of intracellular N. caninum proliferation in rat macrophages, while corresponding bovine GIMAP proteins also restrict parasite growth in bovine cells (Azam et al., 2025) [40] . Together with murine IRG-mediated PVM targeting (Reid et al., 2012) [57] , these findings expand anti-Neospora immunity beyond cytokine production to direct intracellular trafficking and vacuolar targeting. Overall, resistance reflects coordinated innate recognition, IL-12-IFN-γ-driven cellular immunity, metabolic and oxidative restriction, adaptive responses, and direct targeting of the PV, whereas parasite persistence is favored when N. caninum redirects signaling or cel lular processes such as p38 activation and mitophagy to attenuate antimicrobial defenses (Mota et al., 2016; Zhang et al., 2022) [58,39] .

Table 1. Major host immune responses involved in protection against

From host-parasite interaction to neosporosis pathogenesis

The clinical outcome of N. caninum infection reflects the cumulative balance between parasite replication and the host-restriction mechanisms described above. After establishment of the intracellular niche, tachyzoites depend on host resources and organelle interactions to sustain growth. Recruitment of endoplasmic reticulum, mitochondria, Golgi-associated vesicles, lysosomes, and other endomembrane compartments toward the PV, together with acquisition of host-derived cholesterol and sphingolipids, supports parasite replication (Nolan et al., 2015) [34] . At the same time, GIMAP4-, GIMAP5-, and GIMAP6associated recruitment of LAMP1 and PV acidification can render this niche less permissive to growth (Azam et al., 2025) [40] . Disease progression therefore depends not simply on successful invasion but on whether parasite-mediated maintenance of the PV and nutrient acquisition can overcome host mechanisms that destabilize the intracellular compartment. Parasite-intrinsic factors further shape this balance: deletion of NcROP 5 reduces invasion, intracellular replication, cerebral parasite burden, and virulence, whereas loss of NcSAG 1 attenuates disease and decreases brain parasite burdens in offspring (Ma et al., 2017; Abdelbaky et al., 2024) [28,59] . Likewise, deletion of NcGRA 7 or NcROP 40 attenuates disease and reduces maternal brain parasite burdens, with a stronger effect following NcGRA 7 disruption, while loss of NcROP 24 reduces virulence and congenital transmission and impairs parasite growth in bovine macrophages in a host-cell-dependent manner (Rico-San Román et al., 2022; Amieva et al., 2026) [61, 60] . Collectively, these findings show that pathogenicity emerges from interactions between host-cell permissiveness, host restriction, and multiple parasite effectors rather than from a single virulence determinant. Following acute dissemination, host restriction alters parasite distribution and favors establishment of chronic infection. In experimentally infected mice, early infection is characterized by parasitemia and widespread parasite detection in the spleen, liver, and lungs, whereas later infection becomes increasingly concentrated in the brain as peripheral parasite burdens decline (Collantes-Fernández et al., 2006) [62] . A similar CNS tropism is evident in cattle, where later infection is associated predominantly with parasite DNA in neural tissues together with focal necrosis, glial activation, and perivascular inflammation (Nishimura et al., 2013) [63] . Persistence is facilitated by differentiation of rapidly multiplying tachyzoites into slowly replicating bradyzoites associated with tissue cysts. Experimental stress induces bradyzoite-associated markers and cyst-like structures in vitro, although this does not fully reproduce mature tissue-cyst formation (Weiss et al., 1999) [64] . Direct evidence from naturally infected cattle and dogs confirms BAG-positive bradyzoites within tissue cysts, including cysts in skeletal muscle, showing that persistent stages are not confined to the CNS (Peters et al., 2001) [65] . Stage conversion itself may influence virulence: NcBPK 1 deletion increases tachyzoite replication while impairing expression of the bradyzoite marker NcSAG 4 under differentiation conditions, suggesting that differentiation mechanisms help regulate the balance between acute proliferation and chronic persistence (Amieva et al., 2025) [66] . This persistence has major reproductive consequences because chronically infected cows can transmit infection during pregnancy; increased maternal parasitespecific antibody associated with transmission is consistent with pregnancy-associated recrudescence of established infection rather than necessarily requiring a new exogenous exposure (Guy et al., 2001) [67] . Persistent infection therefore provides a reservoir from which recrudescence, renewed dissemination, and endogenous vertical transmission can arise. Pregnancy most clearly demonstrates how parasite replication, tissue tropism, host immunity, and gestational stage converge to determine pathogenesis. During mid-gestation infection, N. caninum reaches the placenta and fetus, producing necrosis and inflammation within maternal caruncular septa and fetal villi and later focal inflammatory and necrotic lesions in the fetal CNS (Maley et al., 2003) [68] . Placental pathology therefore represents both a route of transmission and a major site where parasite replication and host inflammatory responses interact. Early-gestation infection associated with fetal death produces extensive placental necrosis, inflammatory-cell infiltration, and increased expression of IFN-γ, IL-12 p40 , TNF-α, IL-18, IL-2, IL-4, and IL-10, although these responses correlate with rather than establish a direct causal mechanism for abortion (Rosbottom et al., 2008) [69] . Consistently, fetal death after early infection is associated with more extensive parasite dissemination and parasite-associated necrosis in placental and fetal tissues, whereas later infection shows more restricted fetal parasite distribution, particularly toward neural tissues (Gibney et al., 2008) [70] . In contrast, infection at day 210 can produce efficient vertical transmission without abortion, with viable fetuses showing comparatively limited pathology and evidence of improving maternal and fetal control (Benavides et al., 2012) [71] . Parasite genotype further modifies these gestational outcomes. Nc-Spain7 disseminates to placental and fetal tissues more efficiently and is associated with earlier fetal mortality than less virulent Nc-Spain8 (Regidor-Cerrillo et al., 2014) [72] , while low-virulence Nc-Spain1H causes less parasitemia and placental/fetal involvement and did not induce the fetopathy observed with Nc-1 under comparable early-gestation conditions (Rojo-Montejo et al., 2009) [73] . Early infection-dynamics studies similarly show rapid placental colonization, extensive parasite detection, and efficient vertical transmission following Nc-Spain7 challenge, whereas Nc-Spain1H remains markedly more restricted (Jiménez-Pelayo et al., 2019) [74] . Fetal outcome therefore depends not on transplacental infection alone but on the combined effects of gestational age, maternal and fetal immune competence, parasite replication and dissemination, placental injury, and intrinsic parasite virulence. These observations also define major knowledge gaps and potential intervention points. The mechanisms governing progression from intracellular replication to dissemination, stage conversion, chronic persistence, neurological disease, and congenital transmission remain incompletely understood. Functional studies of NcROP 5 , NcSAG 1 , NcGRA 7 , NcROP 40 , NcROP 24 , and NcBPK 1 show that parasite proteins can differentially influence invasion, replication, host-cell interaction, tissue burden, stage conversion, and congenital disease, reinforcing the view that virulence is multifactorial (Ma, J. Liu, et al., 2017; Abdelbaky et al., 2024 [59] ; Rico-San Román et al., 2022 [61] ; Amieva et al., 2025 [66] , 2026). Similarly, although PV-host organelle interactions support lipid acquisition and intracellular replication, it remains unclear which trafficking pathways can be manipulated safely in vivo (Nolan et al., 2015) [34] . Host-directed approaches are attractive because GIMAP-mediated lysosomal recruitment and PV acidification demonstrate that the protected vacuole can be rendered less permissive to parasite growth, although therapeutic manipulation of this response has not been established (Azam et al., 2025) [40] . Parasite-directed approaches are also moving toward functional validation. The CDPK1-targeting bumped kinase inhibitor BKI-1748 inhibits tachyzoite proliferation and disrupts replication and egress in vitro; in pregnant mice, treatment improves offspring survival and markedly reduces vertical transmission, although maternal cerebral parasite burden is not significantly reduced and efficacy in cattle remains unknown (Imhof et al., 2021) [75] . Future studies should therefore integrate parasite genetics, stage-conversion biology, PV-host interactions, placental immunopathology, and host cell-autonomous restriction to identify the mechanisms that determine whether infection progresses toward controlled persistence, neurological disease, abortion, or congenital transmission and to define parasite-or host-directed interventions capable of limiting disease without exacerbating tissue damage.

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76. How to cite this article: Paudel BR, Bhattarai S, Chhetri S, Neupane S, Khati B. Host-parasite interface in Neospora caninum: Intracellular survival, immune restriction, and pathogenesis. International Journal of Veterinary Sciences and Animal Husbandry. 2026;11(10):01-08. doi: https://www.doi.org/10.222 71/veterinary.2026.v11.i10a.3820