Abstract
Background:
Encephalitozoon cuniculi (E. cuniculi) is an obligate intracellular microsporidian parasite with veterinary and zoonotic relevance. Although the polar tube apparatus mediates host cell invasion, the contribution of macrophage-associated receptors and actin-dependent mechanisms to parasite internalization remains unclear.

Aim:
This study aimed to evaluate the contribution of actin polymerization and selected antibody-pretreatment conditions to the formation of parasitophorous vacuoles (PVs) during E. cuniculi infection of J774A.1 murine macrophages.

Methods:
J774A.1 macrophages were infected with E. cuniculi spores at a multiplicity of infection of 10:1. To assess the role of actin-associated uptake, macrophages were treated with cytochalasin D before infection. Microscopybased assays included macrophage stimulation with lipopolysaccharide (LPS), interferon-gamma (IFN-γ), or their combination before infection, as well as pretreatment with Dectin-1 and transferrin receptor 2 antibodies. For gene expression analysis, before infection, macrophages were stimulated with either LPS or IFN-γ. PV formation was evaluated using light microscopy after hematoxylin and eosin staining. The relative expression of inflammatory and receptor-associated genes, including Tnf, H2-Ab1, Ccr2, Ccl3, Tfrc, Tfr2, Tlr2, and Tlr4, was analyzed by real-time quantitative polymerase chain reaction (PCR).

Results:
Actin polymerization inhibition reduced PV formation in infected macrophages, supporting the contribution of actin-associated processes to E. cuniculi intracellular establishment. In the antibody-pretreatment assay, cytochalasin D treatment had a significant main effect on PV formation (p = 0.0047), whereas antibody pretreatment had no significant effect (p = 0.0991), and no significant cytochalasin D × antibody pretreatment interaction was detected (p = 0.7770). Gene-expression analysis showed that E. cuniculi infection alone increased the relative expression of Tnf, H2-Ab1, Ccr2, Tfrc, Tlr2, and Tlr4 compared with unstimulated and uninfected macrophages. The combined LPS + E. cuniculi condition increased Tnf and Ccl3 expression, whereas Tfr2 expression decreased under selected stimulation conditions.

Conclusion:
These findings suggest that E. cuniculi intracellular establishment in J774A.1 macrophages involves actin-associated processes. In contrast, the antibody pretreatment results should be interpreted as exploratory and associative rather than as evidence of functional receptor-specific inhibition. Further studies using validated receptorspecific approaches, isotype-antibody controls, receptor-specific knockdown or knockout strategies, and proteinlevel confirmation are required to define the participation of individual host receptors during E. cuniculi-macrophage interaction.

Key words: Antibody pretreatment; Actin polymerization; Cytochalasin D; Encephalitozoon cuniculi; J774A.1 macrophages; Parasitophorous vacuoles.