Antimicrobial resistance (AMR) is a critical One Health challenge affecting both human and animal health. Tritrichomonas foetus, a protozoan parasite causing reproductive and gastrointestinal disorders in cattle and cats, presents a growing threat due to limited treatment options. While nitroimidazoles such as ronidazole remain the standard of care, their use is restricted in food-producing animals and associated with emerging resistance in feline strains. Propolis, a complex natural resin produced by bees, has demonstrated antimicrobial and antiparasitic activity in other protozoan infections.
Methods
This in vitro study assessed the minimum lethal concentrations (MLC) of ethanolic propolis tincture from the Észak-Alföld region of Hungary against feline- and bovine-derived T. foetus strains, compared to four nitroimidazoles.
Results
Propolis showed promising activity, with an MLC of 1.25 mg/ mL for feline isolates and 0.16 mg/mL for bovine isolates after 48 h. Ronidazole demonstrated reduced efficacy against feline isolates (MLC 32 μg/mL), suggesting partial resistance, whereas bovine isolates remained susceptible (MLC 1 μg/mL).
Discussion
Our findings highlight propolis as a potential alternative treatment for T. foetus, particularly in cattle where nitroimidazole use is prohibited. Standardizing propolis tincture and conducting in vivo studies will be essential to translate these results into clinical applications. This study contributes to efforts to combat AMR and develop sustainable, natural therapeutic alternatives in veterinary medicine, aligning with One Health principles.
Graphical abstract
Graphical abstract illustrating the antiparasitic effects of flavonoid-rich ethanolic propolis tincture against feline Tritrichomonas foetus. The image summarizes the preparation of the hydroalcoholic propolis tincture, the presence of key flavonoids, microscopic observation of motile trophozoites, and the reduction in parasite counts following treatment at various concentrations over 24 and 48 h.
Illustration showing the extraction of flavonoids from a compound using ethanol, resulting in a liquid solution. A molecular structure and three brown objects represent the flavonoids. A microscopic view with yellow circles highlights the presence of cells. A bar chart displays trophozoite counts for feline strains over time, with a specific focus on the two-point-five milligrams per milliliter concentration marked. A protozoan organism is drawn on the right.
Tritrichomonas foetuspropolisantimicrobial resistanceOne Healthfeline trichomonosisbovine trichomoniasisnatural antimicrobialsThe author(s) declare that financial support was received for the research and/or publication of this article. Project no. RRF-2.3.1-21-2022-00001 has been implemented with the support provided by the Recovery and Resilience Facility (RRF), financed under the National Recovery Fund budget estimate, RRF-2.3.1-21 funding scheme.section-at-acceptanceVeterinary Pharmacology and ToxicologyIntroduction
Livestock production is the fastest-growing agricultural sector worldwide (1). However, infectious diseases remain a major challenge, causing direct losses through increased mortality and reduced productivity, pose a significant challenge (2). Furthermore, zoonotic and cross-species diseases pose risks not only to animal populations but also to human health and ecosystem integrity, as highlighted by the One Health framework (3).
Parasitic infections are particularly problematic due to their persistence and limited treatment options. Among protozoans, Tritrichomonas foetus, a monoflagellated parasite (4) is a significant concern, affecting both feline and bovine populations (5–7). Genetic distinctions between the feline and bovine genotypes of T. foetus have been identified, including single nucleotide polymorphisms (SNPs) in the internal transcribed spacer-2 (ITS-2) region and polymorphisms in elongation factor-1-alpha and cysteine protease 8 sequences (4–6, 8).
In domestic cats, T. foetus infections are most common in breeding facilities (9, 10), shelters (11) and exhibition settings (12, 13). Infected cats typically present with gastrointestinal signs such as diarrhea, anorexia, weight loss, abdominal pain, and chronic colitis (14, 15). Metronidazole and tinidazole show limited efficacy (16, 17) and are often unable to fully eradicate the parasite completely (18). Thus, veterinarians often turn to ronidazole (19). Ronidazole is approximately 10 times more effective than metronidazole (20), but is not authorized for cats (21). Despite high doses and prolonged use, ronidazole only achieves a 65% improvement rate against feline T. foetus and can lead to neurotoxic clinical signs in treated animals (14, 15, 20, 22, 23). Consequently, ronidazole is not authorized for use in cats in the European Union (24, 25).
In cattle, T. foetus is a sexually transmitted parasite, prevalent in regions practicing natural insemination (26). Infected bulls act as asymptomatic reservoirs (27), while infected cows may suffer from vaginitis, spontaneous abortion (28), pyometra, and other reproductive disorders, that lead to significant economic losses (26).
Although zoonotic transmission is rare, a documented case in an immunosuppressed patient underscores the potential risk (29). Due to its efficacy, safety, and excellent pharmacokinetic properties, secnidazole has been successfully used in the treatment of human Trichomonas vaginalis (30, 31), and giardiasis in dogs (32) and cats (33). The group of 5-nitroimidazoles remains the primary treatment option for trichomoniasis in human and veterinary medicine (34–39). However, their use in food-producing animals is prohibited (40), leaving cattle infections largely without effective treatment.
The growing antimicrobial resistance (AMR) of protozoans and the limitations of current treatments underscore the urgent need for novel therapeutic strategies (41). The desire for safer, more effective alternatives has driven interest in plant-based extractions, oils (42), antimicrobial peptides (43) and propolis (44).
Propolis is a resinous, bee-derived natural product that has garnered significant attention for its antimicrobial and antiparasitic properties. It has shown promise as a treatment for infection due to the bioactive compounds it contains. Its composition — approximately 50% resin, 30% wax, 10% essential oils, 5% pollen, and 5% organic components (45–49) — is influenced by geographical (50), botanical (45, 46, 51), and climatic factors (52), as well as by seasonal variation (45, 46), and the genetics of the bees (46, 47). Extraction methods also play a critical role in determining propolis efficacy (53). The antimicrobial efficacy of propolis is strongly correlated with its flavonoid and phenolic content, as these compounds are known to disrupt microbial metabolic pathways, inhibit enzyme activity, and induce oxidative stress in pathogens (54–56). Its anti-protozoal effects are thought to involve disruption of phospholipid metabolism, leading to cell lysis (57). Specific components such as rosmarinic acid (58), apigenin (59, 60), resveratrol (61), kaempferol (62), quercetin (63), and caffeic acid (64), can contribute to its efficacy through mechanisms such as increased reactive oxygen species (ROS) production, cytoplasmic vacuolization (65, 66), and inhibition of surface protein complexes (67, 68).
Given these promising properties, this study aimed to evaluate the in vitro efficacy of ethanolic propolis extracts from the Észak-Alföld region of Hungary against T. foetus strains isolated from cats and cattle, in comparison to established nitroimidazole treatments (metronidazole, ronidazole, tinidazole, and secnidazole). We sought to identify propolis as a potential natural alternative for managing T. foetus infections, particularly in light of increasing AMR and the One Health imperative for sustainable and effective therapies.
Materials and methodsPropolis extract preparation
The raw propolis tincture used in this study was sourced from the Northern Great Plain (Észak-Alföld) region of Hungary. The tincture was prepared by combining 1,000 g of propolis with 3,000 mL of 96% ethanol and 1,000 mL of glycerol. A conventional extraction method was employed, wherein the powdered crude propolis was macerated for 3 weeks at room temperature in a sealed, light-protected vessel. The undissolved components were then removed using filter paper (69). The addition of glycerol during extraction enhanced the yield of active ingredients by facilitating a more polar extraction process, as described in the literature (70). A final propolis concentration of 200 mg of raw propolis per mL of solvent, corresponding to the ratio used during tincture preparation (1,000 g propolis in 4,000 mL solvent mixture). Thus, the extract represents a hydroalcoholic propolis tincture containing glycerol as a co-solvent, in line with established preparation methods reported in the literature.
Parasite isolation
The feline strain of T. foetus was isolated from infected cats within a breeding facility in Budapest, Hungary. Samples were collected by a veterinarian for diagnostic purposes (T1–10; samples collected in November 2022) using a transport broth medium (InPouch TF-Feline, Biomed Diagnostics, White City, OR, USA). Despite previous treatment with ronidazole, chronic parasitic carriage persisted in the population. Positive samples were confirmed using polymerase chain reaction (PCR) with the QIAmp DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany). Primary feline cultures were cryopreserved in 10% DMSO in liquid nitrogen at −196 °C for further testing (71).
The bovine strain used was T. foetus (Riedmuller), Wenrich and Emerson, ATCC 30232 (LGC Ltd., Teddington, Middlesex, UK), a reference strain originally isolated from cattle.
Parasite culture
Maintenance and propagation of T. foetus were performed using Trichomonas cysteine peptone liver infusion medium (CPLM; Biolab Zrt., Budapest, Hungary). The medium was autoclaved at 121 °C for 15 min, followed by the addition of 70 mL of sterile inactivated horse serum (Biolab Zrt., Budapest, Hungary). A vial of Trichomonas selective supplement (Biolab Zrt., Budapest, Hungary) was then added, containing 500 mg/vial streptomycin and 80 mg/vial penicillin added to 425 mL of CPLM broth, to inhibit bacterial overgrowth that might outcompete the parasite. Fresh medium was prepared weekly and stored at 4 °C, while cultures were maintained in 15 mL centrifuge tubes at 37 °C under aerobic conditions. The cultures were passaged every 2 days to ensure their growth remained in the log phase.
Parasite morphology was observed using a Leica Microsystems Dmi1 microscope (BioMarker Kft., Gödöllő, Hungary) at 400 × magnification. Quantification of motile trophozoite forms—characterized by their bulb-like shape, undulating membrane, and jerky flagellar movement (Supplementary Video 1)—was conducted using a Bürker chamber (DIN12847, VWR International, LLC., Debrecen, Hungary) and a standard cell-counting formula.
The initial trophozoite count was determined 24 h post-incubation of the received samples. Mean cell counts were calculated across 25 large squares in the chamber. A twofold dilution was achieved by adding 20 μl of sterile isotonic saline to 20 μl of the suspension. The average cell count was then multiplied by the dilution factor and normalized using a factor of 2.5 × 105, yielding the final count in pcs/mL.
Determination of the total phenolic content and total flavonoids content
The total phenolic content (TPC) of the propolis tincture was assessed using the Folin–Ciocalteu method (72) with gallic acid as the standard. For the analysis, 200 μg/mL of the propolis tincture was mixed with 500 μl of Folin–Ciocalteu reagent (10% v/v) and 500 μl of sodium carbonate (2% w/v). The reaction mixture was incubated at room temperature, without light, for 1 h. Absorbance was measured at 700 nm using a Hach DR6000 spectrophotometer (Hach Lange Kft., Budapest, Hungary), with a blank (reaction mixture without propolis tincture) serving as the control. A calibration curve was constructed with standard solutions of gallic acid (0.01–0.5 mM; Merck Life Science Ltd., Budapest, Hungary). The resulting regression equation was y = 0.0061x + 0.0278, with R2 = 0.9987. The TPC results were expressed as gallic acid equivalent (GAE) in milligrams (mg) per gram (g) of the dry weight (DW) of the propolis tincture.
The total flavonoid content (TFC) was determined using the aluminum chloride colorimetric method as described by Dias et al. (73). In this analysis, 125 μl of 1 mg/mL propolis tincture was mixed with 625 μl of distilled water and 37 μl of 5% sodium nitrite solution. After 5 min, 75 μl of 10% aluminum chloride solution was added, followed by 250 μl of 1 M sodium hydroxide and 137 μl of distilled water. The mixture was vortexed thoroughly, and the intensity of the resulting pink coloration was measured at 510 nm using a Hach DR6000 spectrophotometer, with a blank as the control. A calibration curve was constructed with standard solutions of catechol (0.022–1.5 mM; Merck Life Science Ltd., Budapest, Hungary). The resulting regression equation was y = 0.0049x + 0.0152, with R2 = 0.9979. The TFC results were expressed as catechol equivalent (CAE) in mg per g of DW of propolis.
Both TPC and TFC determinations were conducted in triplicate to ensure accuracy and reliability.
LC–MS/MS profiling of selected bioactive compounds
To confirm the accuracy of the colorimetric determinations of total phenolic and flavonoid content, the propolis tincture was additionally analyzed by LC–MS/MS using a SCIEX Exion LC 2.0 UHPLC system (AB Sciex LLC, Framingham, MA, USA) coupled to a SCIEX QTRAP 4500 triple quadrupole mass spectrometer (AB Sciex Pte. Ltd., Singapore) under identical chromatographic conditions. In parallel with the colorimetric assays, the concentrations of three major phenolic constituents of propolis, caffeic acid phenethyl ester (CAPE), pinocembrin, and galangin were quantified based on literature reports of their antimicrobial relevance (74–77). Separation was achieved on a Merck Purospher STAR RP-18 column (150 × 4.6 mm, 3 μm particle size; Merck KGaA, Darmstadt, Germany) maintained at 30 °C. The mobile phases consisted of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid), with a gradient elution from 20 to 80% B over 20 min. The injection volume was 10 μl and the flow rate was 0.8 mL/min. Multiple reaction monitoring (MRM) mode was applied, with compound-specific precursor–product ion transitions optimized for each analyte.
The propolis and nitroimidazole treatment
Parasite growth and viability were assessed following previously described methodologies (78, 79). During the treatment phase, a hydroalcoholic propolis tincture (200 mg/mL in 96% ethanol with glycerol) was employed alongside a control sample. A two-fold dilution series was generated to determine the lowest concentration where the parasites die out. Unlike standard turbidity assessments, this study focused on the change in trophozoite counts across dilutions. A 24-well cell culture plate (VWR International, LLC., Debrecen, Hungary) was utilized. The potential effects of the solvent (ethanol) were also evaluated independently to distinguish the impact of the active compounds in the propolis tincture from that of the solvent.
Each well was filled with 3 mL of CPLM broth, except for the first column. The propolis tincture stock solution (200 mg/mL, well A1) and 96% ethanol-only control (well C1) underwent an initial tenfold dilution (0.3 mL + 2.7 mL), followed by sequential two-fold dilutions across the wells (Table 1). The dilution series for the propolis tincture and ethanol solvent ensured that any observed effects were attributable to propolis’ active compounds rather than the solvent.
Dilution series of starting propolis tincture and ethanol solvent.
Dilution series of 200 mg/mL propolis tincture
Well
A1
A2
A3
A4
A5
A6
Dilution
10×
20×
40×
80×
160×
320×
mg/mL
20
10
5
2.5
1.25
0.62
Well
B1
B2
B3
B4
B5
B6
Dilution
640×
1,280×
2,560×
5,120×
10,240×
20,480×
mg/mL
0.31
0.16
0.08
0.04
0.02
0.01
Dilution series of 96% ethanol solvent
Well
C1
C2
C3
C4
C5
C6
Dilution
10×
20×
40×
80×
160×
320×
%
9.6
4.8
2.4
1.2
0.6
0.3
Well
D1
D2
D3
D4
D5
D6
Dilution
640×
1,280×
2,560×
5,120×
10,240×
20,480×
%
0.15
0.07
0.04
0.02
0.01
0.005
The upper two rows of the plate contained the extracted propolis dilution along with its solvent, while the lower two rows served as controls, containing only the solvent diluted to the same concentrations as those in the upper rows.
The initial parasite suspension was prepared at approximately 2.4 × 105 cells/mL for the feline strain and 5 × 104 cells/mL for the bovine strain, based on proliferation rates consistent with the literature (80–84). Each well was inoculated with 50 μl of the suspension.
Nitroimidazole derivatives stock solutions (ronidazole, metronidazole, tinidazole, and secnidazole) were prepared (Merck KGaA, Darmstadt, Germany) at 1024 μg/mL in DMSO and distilled water (85). A two-fold dilution series was created in CPLM broth across the first two rows of a 24-well plate, with the solvent undergoing an equivalent dilution series. Positive (containing parasites without active compounds) and negative (lacking parasites and active compounds) control plates were included alongside treatment groups. Cultures were incubated at 37 °C under aerobic conditions.
Parasite counts were performed at 24 h and 48 h using a Bürker chamber. Minimum lethal concentrations (MLC) was defined as the lowest concentration at which no motile organisms were observed (79).
Statistical analysis of trophozoite count variations was conducted using the Kruskal-Wallis test in R version 4.3.0 (86, 87). Treatment effects were analyzed across species, strains, treatment durations, and concentrations.
ResultsTotal phenolic and flavonoid content determination
In Hungary, the primary botanical sources of propolis are poplar (Populus spp.) and birch (Betula spp.) buds (47). These sources significantly influence the concentration of phenolic and flavonoid compounds. The total phenolic content (TPC) and total flavonoid content (TFC) were measured dried extract obtained by evaporating the hydroalcoholic propolis tincture. The TPC was determined to be 37.9 ± 0.08 mg gallic acid equivalent (GAE)/g of dried extract, while the TFC was 19.2 ± 0.05 mg catechol equivalent (CAE)/g of dried extract.
Targeted LC–MS/MS quantification of three phenolic constituents with well-documented antimicrobial relevance revealed concentrations of 10.8 ± 0.6 mg/g dried extract for CAPE (Figure 1), 14.6 ± 0.8 mg/g DW for pinocembrin (Figure 2), and 6.1 ± 0.4 mg/g DW for galangin (Figure 3). These compounds together account for 83.1% of the measured TPC, aligning well with values reported for temperate zone propolis. This profile underscores the substantial contribution of these constituents to the tincture’s overall bioactivity.
Multiple reaction monitoring (MRM) chromatogram of caffeic acid phenethyl ester (CAPE) in the propolis tincture. Representative MRM chromatogram of caffeic acid phenethyl ester (CAPE) detected by LC–MS/MS in the ethanolic propolis tincture. The compound was identified using a specific transition with m/z 283 and eluted at a retention time (RT) of 11.2 min. CAPE concentration was quantified at 10.8 ± 0.6 mg/g dried extract. Chromatographic separation was achieved under gradient conditions using a Merck Purospher STAR RP-18 column (150 × 4.6 mm, 3 μm) at 30 °C.
MRM chromatogram of CAPE showing a peak at a retention time of eleven point two minutes. Transition is two hundred eighty-three to one hundred sixty-three with a concentration of ten point eight plus or minus zero point six milligrams per gram dry weight.
Multiple reaction monitoring (MRM) chromatogram of pinocembrin in the propolis tincture. LC–MS/MS MRM detection of pinocembrin (m/z 255) in the analyzed propolis tincture. The compound eluted at 13.1 min and was quantified at 14.6 ± 0.8 mg/g dried extract. The chromatographic method and instrumental parameters were identical to those described for CAPE. The distinct peak confirms the presence and retention behavior of pinocembrin among the main phenolic constituents.
MRM chromatogram of pinocembrin showing a peak at a retention time of thirteen point one minutes. The intensity reaches fourteen arbitrary units. Transition is from two fifty-five to one hundred nineteen, with a concentration of fourteen point six plus or minus zero point eight milligrams per gram dry weight.
Multiple reaction monitoring (MRM) chromatogram of galangin in the propolis tincture. Chromatographic profile of galangin (m/z 269) obtained by LC–MS/MS in MRM mode. The analyte eluted at a retention time of 15.4 min and was present at a concentration of 6.1 ± 0.4 mg/g dried extract. This compound, together with CAPE and pinocembrin, accounts for 83.1% of the total phenolic content (TPC), indicating its strong contribution to the bioactive profile of the propolis tincture.
MRM chromatogram of galangin depicting intensity in arbitrary units against retention time in minutes. A peak at retention time fifteen point four minutes shows details of the transition from two hundred sixty-nine to one hundred fifty-one, with a concentration of six point one plus or minus zero point four milligrams per gram dry weight.
Calibration curves for gallic acid and catechol standards used in the TPC and TFC assays are shown in Figure 4.
Calibration curves for gallic acid and catechol standards used in the determination of total phenolic content (TPC) and total flavonoid content (TFC), respectively. The gallic acid standard curve was constructed over the range of 0.01–0.5 mM, yielding the regression equation y = 0.0061x + 0.0278 with R2 = 0.9987. The catechol standard curve was generated over 0.022–1.5 mM, with the regression equation y = 0.0049x + 0.0152 and R2 = 0.9979. Absorbance measurements were taken at 700 nm (TPC) and 510 nm (TFC), respectively.
Graph showing calibration curves for TPC and TFC standards. The x-axis represents concentration in millimoles per liter, and the y-axis shows absorbance. Gallic Acid (TPC) is plotted with blue circles and Catechol (TFC) with green squares. Both curves show a positive linear trend.Viability and reproduction
Parasite counts were assessed 24 and 48 h after collection, with three parallel treatments performed for each time point. For the feline strain, a marked increase in trophozoite counts was observed after 24 h (138%), followed by a smaller increase by 48 h (8%), culminating in a total increase of 157%. The bovine strain exhibited a similar, but less dramatic rise in trophozoite counts, with an 82% increase in 24 h and an additional 23% increase by 48 h, resulting in a total increase of 124% (Figure 5).
Trophozoite count progression was observed after 24 and 48 h of incubation at 37 °C for feline (A) and bovine (B) strains, relative to the initial parasite count. Data are presented as mean ± standard deviation (n = 3).
Bar charts labeled A and B display the growth of trophozoite counts over time. Chart A shows counts increasing from 7,231,667 at the start to 18,570,000 at forty-eight hours. Chart B shows counts rising from 908,333 at the start to 2,033,333 at forty-eight hours. Error bars represent variability in measurements.Propolis treatment efficacy
Three primary T. foetus cultures from feline origin and three from bovine origin were tested in parallel. For the feline strain, the MLC of propolis was 1.25 mg/mL after 24 and 48 h, corresponding to a 160 × dilution of the initial propolis tincture. For the bovine strain, the MLC was 0.63 mg/mL after 24 h and decreased to 0.16 mg/mL after 48 h, representing 320 × and 1,280 × dilutions, respectively (Figure 6).
Efficacy (mg/mL) of a two-fold dilution series of propolis tincture was evaluated after 24 and 48 h of treatment for feline (A) and bovine (B) strains. The minimum lethal concentration (MLC) was determined microscopically after 24 and 48 h of incubation at 37 °C. Data are presented as mean ± standard deviation (n = 3). For the feline strain (A), the MLC was determined to be 1.25 mg/mL. Further dilutions beyond this point showed increased in the number of parasites, indicating release from the inhibitory effects of the propolis. In the bovine strain (B), the MLC was 0.63 mg/mL after 24 h of treatment, with reduced parasite counts observed at concentrations as low as 0.08 mg/mL. After 48 h of treatment, the MLC decreased to 0.16 mg/mL, with a significant antiparasitic effect persisting down to 0.04 mg/mL. Beyond this dilution, parasites were progressively released from inhibition.
Bar charts labeled A and B show trophozoite counts in pcs/mL at different concentrations of mg/mL. Chart A indicates a higher count compared to Chart B. Each chart displays data for 24 and 48 hours, with starting levels as a reference.
The results indicate that the feline strain was more tolerant to ethanol (9.6%), while the bovine strain exhibited greater sensitivity (4.8%). Treatment duration influenced efficacy in the feline strain, while concentration was the key determinant for the bovine strain. Statistical analysis (p-values) is detailed in Supplementary Table 1.
Nitroimidazole treatments results
Nitroimidazole derivative stock solutions (ronidazole, metronidazole, tinidazole, secnidazole) were prepared with dimethyl sulfoxide (DMSO) and distilled water. DMSO exhibited parasiticidal effects up to 1%, but below this threshold, parasites were released from inhibition. Thus, the observed effects were attributed to the active substances.
For the feline strain, the MLC of ronidazole was 32 μg/mL after 24 h and 16 μg/mL after 48 h. For the bovine strain, the MLC was 1 μg/mL after 24 h and <0.25 μg/mL after 48 h (Figure 7).
The changes in trophozoite counts were assessed after 24 and 48 h of treatment with a two-fold dilution series (μg/mL) of ronidazole for feline (A) and bovine (B) strains. The results indicate that ronidazole demonstrated the highest efficacy in the bovine strain, with a minimum lethal concentration (MLC) of 1 μg/mL, compared to 32 μg/mL in the feline strain. MLC was determined microscopically following 24 and 48 h of incubation at 37 °C. Data are presented as mean ± standard deviation (n = 3).
Bar graphs labeled A and B compare trophozoite counts at various concentrations in micrograms per milliliter. Graph A shows higher counts at lower concentrations and untreated conditions; graph B shows reduced counts, indicating concentration-dependent effects. Measurements include initial counts, 24 hours, and 48 hours, with error bars.
Metronidazole, tinidazole, and secnidazole were tested only on bovine strain. Metronidazole exhibited an MLC of 1 μg/mL at both 24 and 48 h. Similarly, secnidazole demonstrated a consistent MLC of 0.5 μg/mL across both time points Tinidazole’s MLC decreased from 2 μg/mL at 24 h to 0.5 μg/mL at 48 h (Figure 8). The findings demonstrate that secnidazole had the highest efficacy among nitroimidazoles, with a consistent MLC of 0.5 μg/mL, while tinidazole’s efficacy improved with longer treatment duration.
The changes in trophozoite counts after 24 h and 48 h of treatment with a two-based dilution series (μg/mL) of metronidazole (A), tinidazole (B), and secnidazole (C) are depicted for bovine strains. The results are presented as the mean and standard deviation (n = 3). The MLC was evaluated microscopically after 24 h and 48 h of incubation at 37 °C.
Three bar graphs labeled A, B, and C compare trophozoite counts in pieces per milliliter across various concentrations from 512 to untreated, measured at start, 24 hours, and 48 hours. Each graph shows blue and orange bars for 24 hours and 48 hours, respectively, with gray lines indicating start values. Trophozoite counts decrease with increasing concentration in all graphs, with A showing the highest counts at lower concentrations.Discussion
This study evaluated the in vitro efficacy of a naturally derived propolis tincture and various nitroimidazole compounds against T. foetus isolates of feline and bovine origin. Our findings reveal clear differences in susceptibility between the two host-adapted genotypes and highlight the potential of propolis as a natural therapeutic alternative — particularly in the context of AMR and One Health priorities.
CAPE, pinocembrin, and galangin are among the most widely studied phenolic constituents of propolis, known for their potent antimicrobial and anti-inflammatory properties. Their quantification in this study provides additional chemical context for interpreting the observed biological activities. However, propolis is a complex natural product containing over 300 identified compounds, including other flavonoids, phenolic acids, terpenes, and aromatic esters (65, 77). Consequently, while the present targeted profiling captures key bioactive markers, it does not encompass the full chemical diversity of the tincture. Future studies should employ comprehensive untargeted metabolomics to expand the phytochemical coverage and investigate potential synergistic effects among constituents.
The propolis tincture achieved complete eradication of feline-origin T. foetus at a concentration of 1,250 μg/mL. Remarkably, bovine-origin strains demonstrated even higher sensitivity, with effective concentrations dropping to 630 μg/mL after 24 h and further to 160 μg/mL after 48 h. These results align with previous research on the anti-protozoal effects of propolis, though observed variations are likely due to differences in propolis composition, which can be influenced by geographical, botanical, and climatic factors. These findings underscore the need for standardization of propolis tincture — particularly their phenolic and flavonoid profiles — to ensure consistent efficacy and reproducibility in therapeutic applications.
Our results demonstrating the in vitro efficacy of Hungarian propolis against T. foetus are consistent with findings in other protozoan infections. For instance, Freitas et al. reported significant inhibitory effects of propolis on Giardia lamblia trophozoites, with complete eradication at concentrations as low as 500 μg/mL (88). Similarly, Pontin et al. observed substantial antiparasitic activity of Brazilian propolis against Leishmania amazonensis, suggesting a broad-spectrum potential for propolis in protozoan infections (89). These parallels reinforce the relevance of our findings and highlight propolis as a promising natural alternative for managing protozoan pathogens, particularly in the context of emerging antimicrobial resistance.
Nitroimidazoles remain the mainstay of T. foetus treatment, with ronidazole widely used in feline infections. In this study, ronidazole demonstrated an MLC of 32 μg/mL against feline isolates after 24 h, decreasing to 16 μg/mL after 48 h. Given that the resistance threshold is considered >10 μg/mL, this suggests partial resistance in the feline strains we tested (90). This observation is consistent with previous findings reporting MLCs of 1 μg/mL for susceptible strains and up to 100 μg/mL in resistant isolates (20, 91). The suspected resistance in our feline isolates may reflect prior exposure to ronidazole and emphasize the need for diagnostic tools capable of detecting resistant infections to guide appropriate therapy.
In contrast, bovine-origin T. foetus strains exhibited markedly higher susceptibility to ronidazole, with an MLC of 1 μg/mL at 24 h and <0.25 μg/mL at 48 h. Alternative nitroimidazoles (metronidazole, tinidazole, and secnidazole) demonstrated similar or improved efficacy. Notably, secnidazole maintained an MLC of 0.5 μg/mL at both time points, highlighting its potential as an alternative treatment. Tinidazole also showed improved efficacy with prolonged treatment. This marks the first study to evaluate ronidazole’s activity against bovine-origin T. foetus, pointing to promising avenues for further investigation. However, the lack of comparative data on nitroimidazole efficacy against bovine isolates remains a critical gap.
Our findings on the MLC of nitroimidazoles against T. foetus align with existing literature on their efficacy against trichomonas infections. The Centers for Disease Control and Prevention (CDC) notes that metronidazole and tinidazole are the primary treatments for Trichomonas vaginalis, with cure rates ranging from 84 to 98% for metronidazole and 92 to 100% for tinidazole. Secnidazole, a newer nitroimidazole, has also demonstrated high efficacy, with some studies reporting cure rates comparable to or exceeding those of metronidazole and tinidazole (92, 93). These data support our observations of secnidazole’s potent activity against bovine-origin T. foetus strains and suggest its potential as an effective alternative where other nitroimidazoles are less effective or contraindicated.
The role of propolis as an alternative treatment is particularly compelling in light of regulatory restrictions on nitroimidazoles in food-producing animals. Although there are no direct comparative studies on propolis efficacy against feline- or bovine-origin T. foetus, studies in other protozoan infections support its potential. For instance, Brazilian propolis eradicated T. vaginalis at 500 μg/mL (83) while Cuban propolis achieved similar effects at much lower concentrations (3.2–9.1 μg/mL) (94). In contrast, Egyptian propolis required concentrations as high as 75,000 μg/mL against Trichomonas gallinae (95). Hungarian propolis has demonstrated antiparasitic activity in avian studies at concentrations ranging from 1,100 to 5,000 μg/mL (44, 96, 97). Our data suggest that bovine-origin T. foetus is particularly sensitive to propolis, which may be promising for local treatments in breeding bulls and for reducing infection reservoirs in cattle.
Nevertheless, variability in propolis composition remains a major challenge. Standardization of key active compounds — such as flavonoids and phenolic acids — and detailed pharmacokinetic studies are essential to enable reproducible and clinically relevant outcomes. In this study, we observed distinct differences in the sensitivity of feline and bovine T. foetus isolates to both propolis and nitroimidazoles. The partial resistance of feline strains to ronidazole highlights the importance of continued surveillance and alternative therapies to combat AMR. Future studies should prioritize the development of reliable diagnostic assays to differentiate treatment failure due to resistance, reinfection, or suboptimal dosing regimens.
The variability in the effective concentrations of propolis observed in our study aligns with the significant differences reported in other Trichomonas species. For example, a study from Hungary found that ethanolic tincture of propolis from the Észak-Alföld region had a minimum eradication concentration (MEC) ranging from 2.5 to 5 mg/mL against T. gallinae (96). In contrast, an Egyptian study reported that an aqueous propolis tincture required concentrations as high as 50 mg/mL to fully inhibit the growth of T. gallinae within 48 h (95). These findings underscore the critical influence of botanical and geographical factors on the composition and efficacy of propolis, particularly in terms of its flavonoid and phenolic content, and highlight the need for standardization to ensure reproducible and reliable antiparasitic effects.
In summary, our findings contribute to the growing body of evidence supporting natural, sustainable alternatives to conventional antimicrobials in veterinary medicine. By highlighting the potential of propolis and identifying strain-dependent differences in susceptibility, this work aligns with the One Health objective of mitigating AMR while safeguarding animal health and productivity.
Conclusion
This study provides compelling in vitro evidence that Hungarian propolis exhibits significant antiparasitic activity against both feline- and bovine-derived T. foetus strains, with particularly marked efficacy against bovine isolates. These findings are noteworthy given the regulatory restrictions on nitroimidazole use in food-producing animals and the emerging partial resistance of feline strains to ronidazole, underscoring the pressing need for novel, sustainable alternatives in veterinary practice.
Importantly, our results highlight the potential of propolis as a natural antimicrobial agent that aligns with the One Health concept of integrated approaches to combat AMR while preserving animal health and productivity. However, the inherent variability in propolis composition — driven by geographical, botanical, and extraction factors — necessitates standardization of its active constituents to ensure reproducible efficacy and safety. Furthermore, comprehensive in vivo studies, including pharmacokinetic profiling and safety assessments, are essential before propolis can be integrated into therapeutic regimens.
Future research should also prioritize the development of reliable diagnostic tools to detect resistant T. foetus strains, enabling targeted interventions and supporting stewardship efforts to mitigate AMR spread. Collectively, this work contributes to the expanding evidence base supporting the use of natural compounds as adjuncts or alternatives to conventional antimicrobials, offering promising avenues for sustainable disease management in veterinary medicine.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.
Ethics statement
The manuscript presents research on animals that do not require ethical approval for their study.
Author contributions
ÁK: Methodology, Conceptualization, Visualization, Data curation, Formal analysis, Writing – review & editing, Software, Writing – original draft. AY: Writing – original draft, Resources, Conceptualization, Project administration. ÁS: Writing – original draft, Investigation. BT-S: Writing – original draft, Validation. ÁJ: Writing – review & editing, Supervision, Funding acquisition, Validation.
Acknowledgments
Many thanks to everyone who contributed to the study, with special appreciation for the laboratory assistants.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2025.1635358/full#supplementary-material
Live microscopy of motile Tritrichomonas foetus trophozoites observed under phase-contrast illumination (400×). The video illustrates the active flagellar motion and characteristic tumbling behavior of the parasite.
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Edited by: Nora Mestorino, National University of La Plata, Argentina
Reviewed by: Daniel A. Abugri, Alabama State University, United States