Encephalitozoon cuniculi spores (American Type Culture Collection, Manassas, VA, USA, ATCC #50502) were propagated and harvested from Vero (American Type Culture Collection, ATCC CCL-81™) cells as previously described (Han et al., 2019; de Boer et al., 2021). Briefly, Vero cells were grown in T25 flasks containing Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, and maintained at 37 °C in a humidified atmosphere of 5% CO₂ (Barrett et al., 2009; Pollock et al., 2020). Cells were then inoculated with spores at approximately 60% confluence at a multiplicity of infection (MOI) of 10 spores per cell (Guidi-Rontani et al., 1999). The culture medium was replaced every 3 days, and amplified spores were harvested after 10–15 days of culture. Spores were collected from the supernatant, purified by filtration through a 3-μm pore-size membrane to remove host cell debris, and enumerated using a hemocytometer prior to use (Han et al., 2019).

Specific primers targeting the internal transcribed spacer (ITS) region of E. cuniculi were designed (Ghosh and Weiss, 2009; Santín and Fayer, 2011). The process began with retrieving ITS sequences from various Encephalitozoon species from the GenBank database using the NCBI Nucleotide Sequence Search program. Multiple sequence alignments were performed using the Clustal W algorithm within MEGA11 software to identify conserved regions unique to E. cuniculi. Candidate primers were subsequently analyzed in silico with Primer-BLAST to assess species specificity. Further evaluation of primer properties, including melting temperature (Tm), potential for dimerization, self-annealing, hairpin formation, and GC content, was conducted using the PrimerSelect module of DNASTAR software (Table 1). Primer specificity was ultimately confirmed by experimental PCR amplification. The templates used for this validation were high-quality genomic DNA from a panel of organisms, including the closely related Encephalitozoon species E. cuniculi and E. hellem, as well as the common unicellular fungus Saccharomyces cerevisiae and the prokaryotic bacterium Escherichia coli.

In this experiment, we employed two long-acting sporicidal agents: quaternary ammonium compounds (QACs) and nanosilver disinfectant (Ag). The QAC disinfectant (Brand: Basteur®, Shanghai Basteur Antimicrobial Technology Co., Ltd.) contains alkyl dimethyl benzyl ammonium chloride and dioctyl dimethyl ammonium chloride as its active ingredients, at a concentration of 0.18–0.22% (w/v). The nanosilver disinfectant was an aqueous solution of silver nanoparticles (12–15 nm in diameter, 1,000 ppm), supplied by Shanghai Huzheng Nano Technology Co., Ltd. Three rapid-acting disinfectants were evaluated: 75% ethanol (EtOH), a chlorine-based agent (CL), and hydrogen peroxide (H₂O₂). The chlorine-based disinfectant contained sodium dichloroisocyanurate and trichloroisocyanuric acid as its primary components, with an available chlorine content of 45–55%. The hydrogen peroxide solution had a concentration of 0.9–1.1%. All three rapid-acting disinfectants were supplied by Shanghai Basteur Antimicrobial Technology Co., Ltd.

A spore suspension was prepared at two concentrations in PBS: 2 × 10⁷ spores/mL for Vero cell infection and 2 × 10⁸ spores/mL for mouse intraperitoneal injection. Each 1 mL aliquot of the sample suspension was centrifuged at 8,000 × g for 5 min. The resulting pellet was resuspended in 2 mL of one of the five disinfectants. Samples were exposed to rapid-acting disinfectants for 20, 40, or 60 min, or to long-acting disinfectants for 2, 3, or 5 h. After treatment, the samples were centrifuged, washed with sterile PBS, and stored at 4 °C until subsequent use in cellular or animal experiments.

In this study, ethidium bromide (EB) (HY-D0021, MCE China) was chosen to assess sporicidal efficacy. EB is a fluorescent dye with a moderate molecular weight of 394.32, which cannot penetrate spores with intact cell walls. However, when sporicidal agents induce structural damage, EB permeates the compromised cells and intercalates with nucleic acids. Under blue light excitation, damaged spores exhibit distinct red fluorescence (Alakomi et al., 2000; Fedorko et al., 2005). Flow cytometry (Guava EasyCyte, MILLIPORE) was utilized to quantify EB staining in spores before and after treatment. The proportion of spores displaying red fluorescence was analyzed to evaluate sporicidal efficacy, enabling precise quantification of agent-induced cellular damage.

To precisely determine the impact of sporicidal treatments on spore infectivity, spores subjected to different treatments were used to infect Vero cells. At 48 h post-infection, the culture medium was removed from all groups, and extracellular spores were thoroughly washed with PBS. Cells were lysed, and species-specific qPCR using custom-designed TaqMan probes was performed to measure intracellular spores DNA copy number. The sporicidal efficacy was assessed based on the reduction in genomic DNA load, reflecting the ability of treated spores to invade host cells.

Healthy female specific pathogen-free (SPF) C57BL/6 mice (20–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Spores treated with different sporicidal methods were intraperitoneally injected into the mice. On day 45 post-infection, deep anesthesia was induced in the mice using 5% isoflurane delivered in an oxygen stream within an induction chamber. Once a surgical plane of anesthesia was confirmed (absence of response to toe pinch), the mice were immediately euthanized by cervical dislocation (Elsner et al., 2015; Bodnar et al., 2023). Blood and one kidney were collected from each mouse for E. cuniculi DNA quantification by qPCR, while the contralateral kidney was fixed in 4% paraformaldehyde for subsequent fluorescence staining (Sak et al., 2022). For qPCR analysis, blood samples (50 μL) were mixed with 50 μL of one-step lysis buffer (CY-L-001, Shanghai Shuiyuan Biotechnology Co., Ltd.), boiled for 10 min, cooled to room temperature, and stored at −20 °C until use. Similarly, kidney tissue samples (~50 mg) were homogenized in 100 μL of the one-step lysis buffer, boiled for 10 min, cooled to room temperature, and stored at −20 °C. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Southwest University (approval no.: IACUC-20250304-40).

Mouse kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 5 μm. Following deparaffinization and rehydration, the sections were subjected to fluorescence staining. The subsequent staining procedure was nearly identical for both cultured cells and tissue sections. Briefly, samples were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized with 0.05% Triton X-100 for 25 min. After another PBS wash, the samples were stained with PI (HY-D0815, MCE China) and Calcofluor White (HY-D0367, MCE China) at final concentrations of 10 μg/mL and 1 μg/mL, respectively, and incubated at room temperature for 20 min in the dark (Carriere et al., 2023). Finally, the cell samples were washed three times with PBS and mounted in PBS solution containing 50% glycerol for microscopic observation and imaging. The kidney tissue sections were mounted with a synthetic resin mounting medium for microscopic observation.

All experiments were repeated at least three times independently. Data are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) was performed to assess significant differences in variables, followed by Tukey’s post hoc test (SPSS Statistics v. 23.0; IBM Inc., Chicago, IL, USA). Differences were considered statistically significant at p < 0.05.

Using our self-developed species-specific qPCR assay for E. cuniculi, we performed absolute quantification of serially diluted E. cuniculi genomic DNA. Parallel testing included E. hellem (high genomic homology control), yeast genomic DNA (gDNA), and E. coli genomic DNA as amplification templates. Results demonstrated a detection limit of 1.4 fg of genomic DNA, with quantifiable spore DNA copy concentrations as low as 1.359 × 10¹ ± 5.217 copies/μL (n = 3). No amplification was detected for E. hellem gDNA, yeast gDNA, or E. coli genomic DNA (Table 2).

To determine the optimal EB concentration for formal experiments, we prepared serially diluted EB solutions and stained untreated E. cuniculi spore suspensions of uniform concentration. Staining results revealed that normal E. cuniculi spores failed to exhibit fluorescence at concentrations ≤ 0.0625 mg/mL (Figure 1). Consequently, 0.0625 mg/mL was selected for subsequent flow cytometric analysis.

Following the determination of the optimal EB staining concentration, we evaluated three fast-acting disinfectants: 75% ethanol, 1% hydrogen peroxide, and a chlorine-based disinfectant. Disinfection time points were set at 20, 40, and 60 min. Results for 75% ethanol demonstrated a statistically significant increase in the proportion of EB-positive spores compared to untreated controls at 20 min (p < 0.0001). Prolonged exposure beyond 20 min did not further enhance staining efficacy. Notably, during post-treatment centrifugation, the 75% ethanol group exhibited markedly whiter and larger spore pellets—a phenomenon absent in the other disinfectant groups. The chlorine-based disinfectant failed to exhibit significant differences in EB-staining proportions compared to controls across all time points, suggesting ineffective disruption of spore wall integrity. Among the tested agents, hydrogen peroxide showed the most substantial time-dependent increase in EB-positive rates, with 60-min exposure achieving a maximum staining proportion of 62.42 ± 0.34% (Figure 2).

Subsequently, we evaluated two long-acting disinfectants, quaternary ammonium compounds (QACs) and silver ion agents, using the same methods as described above. Compared to rapid-acting disinfectants, the maximum EB-staining rate observed among long-acting agents was 31.76 ± 0.45% in the silver ion group. Notably, post-disinfection centrifugation of QAC-treated samples yielded persistently aggregated pellets that resisted resuspension, with spores forming compact clumps. This aggregation phenomenon potentially compromises the objectivity of flow cytometric assessment for QAC efficacy, as evidenced by characteristic shifts in forward scatter (FSC)/side scatter (SSC) profiles (Figure 3).

To accurately determine whether disinfectant-treated spores retained infectivity, we inoculated Vero cells with spores exposed to various disinfectants. Experiments were conducted in 12-well plates pre-seeded with synchronized Vero cells 24 h prior to inoculation. After 48-h infection, supernatants were meticulously aspirated to eliminate residual extracellular spores, followed by three PBS washes. Cells were lysed thoroughly, and the resultant lysates served as templates for E. cuniculi-specific qPCR. This protocol ensured the exclusive detection of intracellular spores, reflecting functional infectivity. Our findings demonstrated that chlorine-based disinfectants achieved superior infectivity ablation, with > 90% spore infectivity loss within 20 min. Although hydrogen peroxide showed slightly reduced efficacy compared to chlorine-based agents, it eliminated 96% of infectivity after 60-min exposure. In contrast, 75% ethanol (EtOH) exhibited minimal infectivity inactivation, yet showed statistically significant differences from untreated controls at all timepoints (p < 0.001).

Disinfectant efficacy was evaluated using the Log Reduction Value (LRV), defined as the log₁₀ reduction in the mean spore DNA copy number concentration from the untreated control to the disinfected sample. A larger LRV indicates superior efficacy (Rutala et al., 2023). The LRV is formally defined by the equation: LRV = N₀ − Nₓ, where N₀ and Nₓ are the log₁₀-transformed mean spore DNA copy number concentrations for the untreated control and disinfected groups, respectively (Rose et al., 2005; Huang et al., 2022). Consequently, when the post-disinfection mean concentration falls below 1 (making Nₓ ≤ 0), the calculated LRV will be greater than or equal to N₀ (Table 3).

To visually assess infectivity, spores subjected to various disinfectant treatments were recovered, washed three times with PBS, centrifuged, and finally resuspended in complete medium for cell infection.After 48 h, the supernatant was removed, and the cells were washed with PBS, followed by double staining with propidium iodide (PI) and Calcofluor White M2R—a dye that binds to polysaccharides such as cellulose (Green et al., 2000). Among the different treatment groups, the most intense Calcofluor White M2R staining was observed in the control group, followed by the ethanol group, while the chlorine-based disinfectant group exhibited the weakest staining (see Figure 4).

To validate the efficacy of disinfectants against E. cuniculi in vivo, spore suspensions were adjusted to 10⁸ spores per aliquot and treated with various long-acting and short-acting disinfectants. The control group underwent the same centrifugation and recovery procedures but without any disinfectant exposure. All mice were euthanized 45 days post-intraperitoneal injection, and blood and kidney tissues were collected for E. cuniculi DNA detection (Sak et al., 2020). Untreated control mice showed E. cuniculi DNA levels of approximately 10⁴ copies/μL in both kidneys and blood. In contrast, all disinfectant-treated groups exhibited markedly reduced or undetectable levels of E. cuniculi DNA. Notably, no E. cuniculi DNA was detected in either blood or kidney samples from mice exposed to the long-acting quaternary ammonium compound (QACs, 5-h treatment) or the short-acting disinfectants (chlorine-based agent and hydrogen peroxide, 1-h treatment). Conversely, 75% ethanol demonstrated the least efficacy among the tested disinfectants, with detectable E. cuniculi DNA persisting in both kidneys and blood of mice after either 20-min or 1-h treatments (Table 4).

The kidney is a primary target organ of E. cuniculi infection (Rodríguez-Tovar et al., 2016). To corroborate the quantitative PCR (qPCR) results from disinfected tissue samples, kidney tissues from each experimental group were fixed, embedded, and sectioned. The rehydrated sections were subjected to fluorescence staining with Calcofluor White (CFW) and propidium iodide (PI). PI staining, which emits red fluorescence, was used to delineate tissue structure. Since the inner layer of the microsporidian spore wall is primarily composed of chitin, spores are an ideal target for CFW, which produces intense, specific blue–green fluorescence upon binding under ultraviolet light.

In the present study, kidney sections from untreated control mice exhibited strong blue CFW fluorescence signals, primarily localized to the walls of small blood vessels, the tubular basement membrane, and the pelvi-ureteric junction. In contrast, the CFW signal in kidney tissues from disinfectant-treated infected mice was markedly less intense and extensive compared to that in the control group. Notably, virtually no detectable CFW signal was observed in groups treated with the chlorine-based disinfectant for 1 h, hydrogen peroxide for 1 h, or the quaternary ammonium compound (QACs) for 5 h (Figure 5).