Search Results (1,670)

Background/Objectives: Influenza A (IAV) and influenza B (IBV) viruses pose significant public health threats, with varying epidemiology and immune responses. Limited subtype-specific cytokine data exist for influenza in Saudi Arabia. This study conducted molecular surveillance on 380 NPAs from patients at King Khalid University Hospital (KKUH) in Riyadh, Saudi Arabia, during winter seasons (2020–2023). Methods: NPA samples were collected from hospitalized patients presenting with fever (>38 °C) and respiratory symptoms. RNA was extracted using the QIAamp Viral RNA Kit, followed by RT-PCR for IAV (H1N1, A/H3N2) and IBV detection. Quantitative real-time PCR profiled mRNA expression of 17 cytokines/chemokines in IAV-positive (n = 65) and IBV-positive (n = 20) samples, normalized to GAPDH using the 2^−ΔΔCq method. Appropriate statistical tests were applied (p < 0.05 significant). Results: Results showed 17.11% IAV positivity (7.89% A/H1N1, 9.21% A/H3N2) and 5.26% IBV. A/H3N2 predominated, increasing from 6.67% (2020/21) to 12.30% (2022/23). Males had higher IAV rates (25.88% vs. 10.00% females, p < 0.05), while IBV was higher in females (6.67% vs. 3.53%). Age-wise, 0–4 years had peak IAV (28.42%, p < 0.05); IBV peaked at 5–14 years (10.91%). IAV elicited higher mRNA expression IFN-α, IL-10, IL-13, and CCL-2 (p < 0.05); IBV showed elevated IL-1α, IL-6, and IL-33 (p < 0.05). Within IAV, A/H1N1 had higher IL-4, IL-10, IL-13, and IL-17; A/H3N2 elevated TNF-α, IL-6, IL-22, CCL-3, and CCL-4 (p < 0.05). Conclusions: These findings highlight subtype-specific inflammatory profiles and demographic disparities in Saudi Arabia, informing targeted interventions. Post-COVID resurgence underscores surveillance needs amid travel and gatherings. Insights into cytokine dynamics aid prognosis and therapeutics, emphasizing regional molecular monitoring for vaccine optimization and outbreak prevention. Full article

This study aimed primarily to evaluate the performance of two Conformité Européenne—In Vitro Diagnostic (CE-IVD) multiplex real-time PCR (rt-PCR) assays for the molecular identification of tick-borne pathogens (TBPs) of human interest on ticks removed from human skin and collected through a citizen science-based approach. As a secondary objective, the aggregated results were used to describe tick species distribution, developmental stages, and seasonal TBP circulation in 2024 in the considered area. The comparison was conducted on 116 tick samples collected in 2024 voluntarily delivered to a hospital in northeastern Italy. Detected TBPs were further confirmed with in-house-validated PCR methods and, where applicable, resolved to the species level. Clinically relevant pathogen species were identified as single infections or coinfections. Overall, 33.6% of tick samples tested positive for at least one TBP, and 6.9% showed coinfections. Kit B exhibited a higher detection rate for Borrelia spp. and Rickettsia spp. targets, partly reflecting its broader diagnostic specificity, while statistically significant differences in cycle threshold values were observed for Anaplasma phagocytophilum detection. The most frequently involved ticks were Ixodes ricinus nymphs, and the most represented area was Verona province. Late spring and early summer were identified as the periods with the highest tick conferment and pathogen diversity. Overall, the results support the use of multiplex real-time PCR commercial kits combined with citizen science-based tick collection as an effective approach for both diagnostic screening and regional surveillance of circulating ticks and TBPs. Full article

Fluorine-free paper coatings with water- and oil-resistance properties have gained considerable attention for sustainable food packaging applications. In this study, a dual-layer coating based on chitosan (Chi) and acrylated epoxidized soybean oil (AESO), both derived from renewable and natural resources, was applied to kraft paper. The ultraviolet-cured AESO top layer formed a dense crosslinking network, while the Chi interlayer promoted strong interfacial adhesion with the kraft paper through hydrogen bonding, effectively restricting fluid penetration. The Chi/AESO₄₀/kraft paper showed markedly enhanced water repellency and oil resistance, with a reduced Cobb₆₀₀ value of 16 g m⁻² and kit rating of 12. Thermogravimetric analysis demonstrated improved thermal stability, and mechanical testing results revealed enhanced packaging-relevant strength, with the tensile strength increasing from 33 to 40 MPa and tensile index increasing from 45 to 60 kPa·m² g⁻¹; furthermore, the burst strength and index improved from 260 to 330 kPa and from 3.2 to 4.0 kPa·m² g⁻¹, respectively. Food contact tests conducted using French fries confirmed the effective barrier performance of the Chi/AESO/kraft paper, highlighting its potential for use in sustainable paper-based food packaging applications. Full article

(1) Background: The oral cavity is a complex ecological environment that integrates elements of both the digestive and respiratory systems, contributing to its extensive microbial diversity. Despite its effectiveness, chlorhexidine is associated with undesirable effects, such as mucosal irritation and tooth staining, which have prompted research into natural alternatives. This study aimed to compare the antimicrobial activity of mouthwashes containing tea tree oil (TTO) alone and in combination with cannabidiol (CBD) and spilanthol with that of a chlorhexidine digluconate-based mouthwash (CHX) against selected oral bacterial and fungal strains. (2) Methods: To assess the antimicrobial effects of the tested mouthwashes on reference microbial strains, the agar diffusion method was applied in accordance with the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). In addition, the microdilution method using 96-well microtiter plates was employed to determine the minimum inhibitory concentration (MIC) of the tested substances. Microbial viability was further evaluated using the WST-based colorimetric Microbial Viability Assay Kit, in which the intensity of the produced WST-formazan dye is directly proportional to the number of viable cells. (3) Results: In the disc diffusion assay, inhibition zones measured after 24 h varied among the tested microorganisms, with the largest zones observed for CHX against Candida parapsilosis (19.63 mm) and Streptococcus pyogenes (16.7 mm). In the microdilution assay against Candida albicans, the MIC₅₀ was achieved for preparations A and B at the highest tested concentrations (column 1), whereas for chlorhexidine (CHX), it was reached at lower concentrations (column 9). (4) Conclusions: ① All tested mouthwashes containing tea tree oil (TTO), either as a single active ingredient or in combination with cannabidiol (CBD) and spilanthol, demonstrated limited bacteriostatic and antifungal activity under the experimental conditions of this study. ② The chlorhexidine digluconate-based mouthwash exhibited significantly higher antibacterial and antifungal activity against all tested microbial strains compared to both the TTO-only mouthwashes and the formulation containing TTO combined with CBD and spilanthol. Full article

Partial oxidation of methane (POM) is a good way to make syngas because it uses exothermic reactions to keep itself going. This study made a series of Ni/KIT-6 catalyst precursors with Gd (0.5–2 wt.%) added to them and then carefully looked at how they changed into active catalysts. The first tests on the precursors using N₂ physisorption, XRD, and H₂-TPR showed that they had a high surface area and changed how they reduced. However, the high-temperature activation (700 °C) and reaction (682 °C) conditions caused thermal evolution and sintering. Tests of catalytic performance and RSM optimization found that the 5Ni + 1Gd/KIT-6 formulation was the best. Under the best conditions, it converted 89.0% of CH₄ and 87.4% of H₂. Using TEM and Raman spectroscopy to look at the used catalysts showed that 1 wt.% Gd was able to control the size distribution of the metallic particles and stop disordered carbon from forming, even after thermal recrystallisation. A 24 h stability test confirmed these findings, indicating a stable H₂ yield (85–87%) and minimal performance degradation, thereby demonstrating that Gd promotion maintains the stability of the active metallic phase under operational stress. Full article

Background/Objectives: Saliva plays an essential role in maintaining the oral ecological balance, and its quantitative and qualitative characteristics may influence susceptibility to dental caries. The aim of this study was to determine susceptibility to dental caries based on the DMFT index and to establish a correlation between caries experience and salivary parameters in a group of young adults. Methods: This cross-sectional study was conducted between July and November 2025 on a sample of 87 fourth-year students from the Faculty of Dentistry in Craiova. Each participant underwent an intraoral clinical examination to determine the DMFT index. The salivary parameters assessed included unstimulated salivary flow rate, saliva consistency, salivary pH, stimulated salivary flow rate, and buffering capacity, using the GC Saliva-Check Buffer kit. Statistical analyses were performed using SPSS (Statistical Package for Social Sciences) software, version 26 (SPSS Inc., Armonk, NY, USA). Results: The mean DMFT index value for the entire sample was 8.26 ± 4.481, with higher values observed among female participants. Low salivary pH was significantly associated with higher DMFT values. Participants with low or very low buffering capacity exhibited higher DMFT values compared to those with normal capacity, indicating that a reduced ability to neutralize salivary acidity is associated with increased caries activity. Conclusions: The results indicate that salivary pH and buffering capacity are important factors in dental caries susceptibility among young adults. The integration of salivary testing into the diagnostic assessment of caries risk may contribute to personalized and effective preventive strategies. Full article

According to the EFSA Report on Zoonoses (2024), yersiniosis was classified as the fourth most commonly reported zoonosis in humans in 2023, with a 13.5% increase in yersiniosis infections compared to 2022. In 2024, the findings were consistent with the 2020–2023 trend. Isolation and identification of enteropathogenic Yersinia is difficult and time consuming, especially when examining food and environmental samples. Among them, Y. pseudoturbeculosis poses a challenge due to the lack of a single selective medium for all bioserotypes. Therefore, faster methods for the detection of Yersinia spp. need to be implemented into the praxis. Rapid identification of pathogens in food or at the time and location of the epidemiological outbreak (point-of-care testing) enables either prevention of the outbreak or early stage diagnosis and prompt decisions. The loop-mediated isothermal amplification (LAMP) is increasingly coming to scientists’ attention as a robust and rapid methodology for pathogen detection in laboratories with limited resources and equipment. The aim of current study is to evaluate, for the first time, the sensitivity of the LAMP protocol based on colorimetric detection in the visible spectrum in comparison with that of the digital droplet PCR (ddPCR). For this aim, a series of decimal logarithmic dilutions of the pathogen Y. pseudotuberculosis in artificially contaminated raw goat milk was used. One commercial LAMP kit with two different dyes (one dsDNA-binding and one Mg²⁺-sensitive) was compared to the sensitivity of the detection to ddPCR. The results obtained revealed a high sensitivity of the kit for detection of DNA isolated from artificially contaminated milk samples in the following range: visible detection based on visible color change—3.1 × 10⁴ mL (violet dye) and 3.4 × 10³/mL (blue dye); detection with gel electrophoresis—2.0 × 10¹/mL (violet dye) and 3.4 × 10²/mL (blue dye). The enumeration of the DNA copies in the same samples was performed with ddPCR, with a detection limit of 2.0 × 10¹/mL. Our results indicate the potential and the possible applicability of the LAMP method for rapid and sensitive visual detection of Y. pseudotuberculosis in raw goat milk. The presented ddPCR protocol can be used for highly sensitive identification and enumeration of Y. pseudtuberculosis in raw goat milk. In conclusion, the conducted comparison is of importance for future implementation of LAMP protocols for on-field analysis near the sampling site and point-of-care or laboratory diagnostics of Y. pseudtuberculosis after the successful validation procedure of an appropriate LAMP protocol. Full article

Background/Objectives: Rapid diagnostic tests (RDTs) for human immunodeficiency virus (HIV) are widely used, but most kits lack standardized internal quality control (IQC) materials. In this study, we aimed to develop and evaluate a plasma-based IQC compatible with five HIV RDT brands and with proven long-term stability. Methods: Control samples at three reactivity levels were tested with five HIV RDT kits in lyophilized and liquid forms. Lyophilized samples were produced with and without trehalose, whereas liquid samples were prepared with and without StabilZyme™ SELECT Stabilizer (Stabilizer). Accelerated stability testing was performed at 37 °C and 45 °C for 28 days, and the most stable formulation was selected for long-term storage at 4 ± 2 °C and 25 ± 5 °C. Stability was assessed based on test-line visibility and signal intensity. Signal-intensity trends were analyzed using simple linear regression with a t-test on the slope; samples were considered stable when no significant trend was detected (p > 0.05). Results: Reactivity measured using the Elecsys HIV combi PT assay yielded cutoff index (COI) values of 772.65 (1:8) for the strong-positive control and 269.95 (1:25) for the weak-positive control. Trehalose-containing lyophilized samples maintained reactivity under accelerated testing at 37 and 45 °C and for 6 months at 4 ± 2 °C and 25 ± 5 °C, with no significant change in signal intensity (p > 0.05). Conclusions: The plasma-based IQC materials were compatible with all five HIV RDTs, and trehalose-stabilized lyophilized plasma showed high stability, supporting transport and storage without strict cold-chain requirements. Full article

Background/Objectives: To overcome bottlenecks in the application of Macleaya cordata extract (MCE) in veterinary traditional Chinese medicine, such as low bioavailability of its active ingredients, gastrointestinal irritation, and muscular toxicity, this study aimed to develop a liposomal nano-delivery system loaded with MCE (MCE-Lips) to achieve the core objective of “enhancing efficacy and reducing toxicity” and to explore its potential application and mechanism of action in treating bacterial diarrhea. Methods: MCE-Lips were prepared using the thin-film dispersion method, and their physicochemical properties—particle size, encapsulation efficiency, and drug loading capacity—were characterized. In vitro, cytotoxicity against skeletal muscle cells and NCM460 intestinal epithelial cells was evaluated using the CCK-8 assay. The release of lactate dehydrogenase (LDH) from skeletal muscle cells was measured with an LDH assay kit. The expression levels of inflammatory factors (TNF-α, IL-6, and IL-1β) in both cell types were determined through ELISA. A fluorescent probe was employed to assess cell membrane integrity. The effect of MCE-Lips on the expression of tight junction proteins (ZO-1, Occludin, and Claudin-5) was evaluated via immunofluorescence. Acute toxicity was examined through H&E staining. A bacterial diarrhea model was established using Escherichia coli in mice, and comprehensive safety and efficacy were assessed through hematological tests and gastrointestinal motility evaluation. Finally, untargeted metabolomics and 16S rRNA sequencing were utilized to investigate the underlying mechanisms of action. Results: The prepared MCE-Lips had an average particle size of 86.49 nm and a high encapsulation efficiency of 89.07%. In vitro experiments demonstrated that MCE-Lips significantly alleviated skeletal muscle cell damage, reduced LDH release (p < 0.05), and effectively inhibited the expression of inflammatory factors IL-6, TNF-α, and IL-1β (p < 0.05). In NCM460 cells, MCE-Lips exhibited a more pronounced inhibitory effect on LPS-induced release of TNF-α (p < 0.01), IL-6 (p < 0.0001), and IL-1β (p < 0.0001) and enhanced intestinal barrier function by upregulating the expression of tight junction proteins ZO-1 (p < 0.001), Occludin (p < 0.01), and Claudin-5 (p < 0.01). In the bacterial diarrhea model, MCE-Lips showed excellent anti-diarrheal efficacy (p < 0.01). Hematological analysis indicated no systemic toxicity. At the endocrine level, the high-dose group significantly reduced motilin (MTL) levels (p < 0.01), which slowed intestinal motility and prolonged chyme retention, thereby alleviating diarrhea symptoms. Mechanistic studies revealed that it acts by regulating the intestinal metabolic profile and microbial community structure, with Desulfovibrio, Enterococcus, and Streptococcus identified as key characteristic differential genera. Conclusions: For the first time, an MCE liposome nanoparticle system was constructed, and untargeted metabolomics combined with 16S rRNA sequencing were employed to elucidate its anti-diarrheal mechanism. MCE-Lips exerts excellent antibacterial diarrhea effects through multiple mechanisms, including direct cytoprotection and anti-inflammatory action, enhancement of the intestinal barrier, regulation of gut function, and remodeling of the gut microecology. This work provides a novel paradigm for plant-derived nano-anti-diarrheal agents. The systematic evaluation of the pharmacodynamics of MCE-Lips in a piglet bacterial diarrhea model will lay a solid foundation for its eventual market application. Full article

Background/objectives: A rise in infections associated with carbapenem-resistant Providencia species (CRPS) has been observed worldwide. This study presents a genomic analysis of CRPS isolates from four hospitals in Croatia and the outpatient setting, in order to determine the extent of the spread of CRPS in Croatia. In the present study, we applied a combination of phenotypic characterization and molecular analysis of resistance traits to determine the mechanisms and the routes of spread of CRPS. Material and methods: The antibiotic susceptibility testing was performed using disk-diffusion and broth dilution methods. The nature of extended-spectrum β-lactamases (ESBLs), carbapenemases, and fluoroquinolone resistance determinants was investigated by polymerase chain reaction (PCR). In order to obtain an insight into the whole resistome, selected isolates were subjected to the Interarray Genotyping Kit CarbaResist and whole genome sequencing (WGS). Results: In total, 30 isolates were collected from four centers, located in different geographic regions of Croatia. There was uniform resistance to piperacillin-tazobactam, cefuroxime, expanded-spectrum cephalosporins (ESCs), imipenem, ertapenem, meropenem, and ciprofloxacin. Immunochromatographic testing and PCR revealed OXA-48 and NDM carbapenemase in 15 isolates, respectively. Phenotypic tests for ESBLs were positive in all OXA-48 and one NDM-positive organism (16 isolates). The isolates were categorized as extensively drug-resistant (XDR). OXA-48-producing isolates were susceptible only to ceftazidime-avibactam, whereas NDM producers were susceptible to cefiderocol and, in the majority of cases, also to amikacin. WGS identified a plethora of genes encoding resistance to aminoglycosides, such as aadA1 and aadA2, (aph(3″)-Ib and aph(6)-Id, sulfonamides sul1 and sul2, trimethoprim dfrA1, dfrA10, and dfrA12, tetracyclines tet(A) and tet(B), and chloramphenicol catA3 and catA5. Conclusions: Providencia spp., in spite of being a rare pathogen, should be included in the surveillance studies across the medical centers in Croatia. Full article

Background/Objectives: Taiwan cinnamon leaves have been reported to be effective in improving chronic diseases. Herein, cinnamon leaf extract (CLE) and nanoemulsion (CLEN) were prepared to explore their effects in improving sexual dysfunction in rats. Methods: Following extraction with 80% ethanol and analysis by UPLC-MS/MS, CLEN was prepared using an optimal ratio of soybean oil, lecithin, Tween 80, deionized water, and CLE. A total of 48 male rats and 48 female rats were used, with the former being induced with erectile dysfunction, followed by treatment with CLEN or CLE at two doses (100 mg/kg and 50 mg/kg) for 4 weeks. After conducting the penile reflex test, male rats were paired with female rats for measurement of sexual behavior and ICP/MAP. Following sacrifice, α-SMA, nNOS, and β-III tubulin expression areas were measured by histochemical analyses; SMC/collagen ratio by Masson’s trichrome staining; and NO, cGMP, and PDE5 levels by ELISA kits. Results: CLEN was more effective than CLE in increasing intromission frequency, decreasing intromission and ejaculation latency, and recovering erectile response for improving copulatory and ejaculatory performances. A higher maximum ICP/MAP ratio was shown for CLEN through elevation of neurovascular function and erectile capacity. Additionally, CLEN efficiently reduced fibrosis, enhanced neuronal marker expression, and increased the SMC/collagen ratio, leading to penile tissue protection and neural regeneration. Both treatments showed elevated levels of NO and cGMP with a reduction in PDE5, probably through modulation of the NO-cGMP signaling pathway. Conclusions: CLEN was more effective than CLE in restoring erectile function in rats. Some more clinical trials are needed to verify this finding. Full article

Background/Objectives: Dental anxiety is a common clinical problem that negatively affects patient cooperation, treatment acceptance, and physiological stability during dental procedures. This randomized controlled clinical trial study aimed to evaluate the effectiveness of music-based interventions in reducing dental anxiety and stress responses during restorative dental treatment. The null hypothesis was that music exposure would not result in significant differences in anxiety levels or physiological stress parameters compared with standard dental care. Methods: Seventy-five patients with moderate to high pre-treatment dental anxiety (MDAS ≥10) were randomly assigned to three groups: classical music, Turkish music, and control (no music) (n = 25 per group). Anxiety levels were assessed using the Modified Dental Anxiety Scale (MDAS). Restorations were performed using a standardized adhesive protocol. Physiological parameters, including systolic and diastolic blood pressure (SBP, DBP), heart rate (HR), and oxygen saturation (SpO₂), as well as salivary cortisol and alpha-amylase levels, were measured before and after restorative treatment. Salivary cortisol and amylase levels were measured using a Human ELISA Kit. Statistical analysis was performed using paired t-tests and one-way ANOVA with Tukey’s post hoc test (p < 0.05). Results: Both music groups showed significant reductions in SBP, DBP, HR, cortisol, amylase, and MDAS scores compared to the control group (p < 0.05). Oxygen saturation increased significantly in the music groups, whereas it decreased significantly in the control group. There were no significant differences between classical and Turkish music regarding their anxiety-reducing effects. Conclusions: Music-based interventions effectively reduce dental anxiety and physiological stress during restorative dental procedures. This study is novel in simultaneously evaluating subjective anxiety scores and multiple physiological and biochemical stress markers in adult patients undergoing restorative treatment, supporting music as a simple and non-invasive adjunct in clinical dentistry. Full article

Commercial immunoassay-based test kits are widely used for rapid screening of deoxynivalenol (DON) in maize; however, inconsistent results are frequently observed under commercial testing conditions. This study evaluated two distinct contributors to such variability: analytical cross-reactivity of commercial DON immunoassays and between-site variability arising from routine grain elevator testing practices. Under controlled laboratory conditions, all kits accurately measured DON but responded differently to co-occurring DON derivatives. In naturally contaminated maize, immunoassay results reflected the combined presence of DON and co-occurring derivatives, consistent with differences in antibody specificity. An interlaboratory comparison involving multiple grain elevators analyzing identical blinded samples demonstrated substantial between-site variability in reported DON concentrations, with about 16% of results deviating by more than ±20% from the LC–MS/MS reference value. Collectively, these findings show that inconsistent DON test outcomes arise from the combined effects of antibody cross-reactivity and site-specific testing variability, rather than from any unreliability of the analytical methods themselves. This finding highlights the importance of interpreting rapid DON measurements considering these factors. Full article

The drug-encapsulated hybrid hydrogels possessed several expected properties, including porous microstructure, conductivity, adhesive strength, antioxidant activity, antibacterial properties, and cytocompatibility, and have great potential in biomedical applications, such as skin wound hydrogel dressings and bio-adhesives. In this paper, the quercetin-loaded hybrid hydrogels (SSA-QRs) were fabricated using silk fibroin (SF), alginate, and silver-doped reduced graphene oxide (Ag@rGO) nanosheets, incorporating quercetin-encapsulated PF-127 (PF127-QR) micelles. Scanning electron microscopy (SEM) images confirmed that the fabricated hybrid hydrogels possessed an interconnected porous microstructure. The mechanical properties of hydrogels could be regulated by adjusting the content of incorporated Ag@rGO nanosheets and PF127-QR micelles. Furthermore, the obtained SSA-QR hydrogels displayed the expected swelling properties, and the swelling rates could reach 1200–1700% in 120 min, in the equilibrium state. The fabricated SSA-QR hydrogels possessed apparent conductivity and self-healing ability. In addition, SSA-QR hydrogels exhibited strong adhesive performance on the surface of different materials, including skin, metal, wood, plastic, and glass. The typical antibacterial testing using Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) confirmed the excellent antibacterial activities of SSA-QR hydrogels. Moreover, SSA-QR hydrogels displayed good antioxidant ability and intracellular ROS scavenging ability. However, the increased content of Ag@rGO nanosheets could cause a great increase in the hemolysis ratio for SSA-QR hydrogels. Fluorescent images, cell counting kit-8 (CCK-8) assay, and cell scratch testing confirmed their excellent cytocompatibility and cell pro-migration ability. The available results demonstrated a facile strategy to prepare the quercetin-loaded hydrogel for applications of wound hydrogel dressing and bio-adhesives. Full article

Toxoplasma gondii is a globally distributed zoonotic parasite that infects a wide range of warm-blooded animals and poses serious threats to many endangered species. Effective serological detection is critical for disease surveillance and control, yet the accuracy of commercial assays varies significantly across host species. Validated serological methods for the Chinese red panda (Ailurus styani) remain scarce, limiting effective monitoring and conservation efforts. A total of 57 serum samples were collected from captive red pandas. Three commercial serological kits—the modified agglutination test (MAT), indirect hemagglutination assay (IHA), and enzyme-linked immunosorbent assay (ELISA)—were used to detect T. gondii antibodies. Statistical analyses included agreement assessment (Cohen’s kappa), McNemar’s test, and receiver operating characteristic (ROC) curve analysis, with MAT as the reference standard. MAT detected T. gondii antibodies in 36.84% (21/57; 95% CI: 24.32–49.36) of samples, while ELISA identified 42.11% (24/57; 95% CI: 29.29–54.92). IHA failed to detect any positive samples (0%). MAT and ELISA showed almost perfect agreement (κ = 0.817; 95% CI: 0.66–0.97) with no significant difference (p > 0.05). Compared to MAT, ELISA exhibited 95.2% sensitivity and 88.9% specificity. The combined MAT/ELISA approach with retesting of discordant samples yielded a final seroprevalence of 35.09% (20/57), with 8.77% (5/57) indeterminate results. MAT and ELISA are reliable methods for serological surveillance of T. gondii in red pandas, while IHA is unsuitable. We recommend MAT for initial screening followed by ELISA confirmation. The high seroprevalence highlights the need for integrated control strategies, including management of stray felids and rodents in captive environments to reduce transmission risks. Full article