Fructooligosaccharides modulate intestinal fermentation and immune response during Giardia lamblia infection in Meriones unguiculatus Abstract
Giardia lamblia infection alters gut physiology and microbiota interactions, but the impact of prebiotic supplementation in this context remains unclear. This study investigated how fructooligosaccharide (FOS) treatment affects microbial fermentation, intestinal function, inflammation, and hepatic bile acid synthesis in G. lamblia-infected gerbils. Gerbils were divided into four groups: uninfected control, infected control, uninfected and FOS-treated, and infected and FOS-treated. Cecal short-chain fatty acids (SCFAs), intestinal disaccharidase activities, serum and intestinal cytokines, and hepatic expression of Cyp7a1 and Cyp8b1 were analysed. Giardia infection increased cecal acetate, reduced butyrate, elevated serum TNF-α, and increased hepatic Cyp7a1 expression. FOS supplementation increased cecal propionate and other SCFAs and elevated IL-10 levels in serum and intestinal tissue. Notably, FOS reduced intestinal maltase activity regardless of infection status, without affecting lactase activity. Together, these data indicate that giardiasis alters microbial fermentation and inflammatory responses, whereas FOS supplementation primarily promotes a more regulatory immune profile, characterized by increased IL-10 levels, alongside shifts in microbial fermentation, without directly modifying hepatic bile acid–related gene expression. These findings highlight complex diet–microbiota–host interactions during intestinal parasitic infection and support further mechanistic studies.
Key words
Fructooligosaccharides; Giardia lamblia; Short-chain fatty acids; Bile acid metabolism; Meriones unguiculatus
INTRODUCTION
Giardia lamblia is a flagellated protozoan parasite that colonizes the small intestine and causes giardiasis, a globally prevalent enteric disease marked by diarrhea, malabsorption, and mucosal inflammation (Allain et al. 2021). Beyond direct epithelial disruption, Giardia infection alters gut microbiota composition and impairs host–microbe interactions, with implications for nutrient metabolism, intestinal immunity, and systemic homeostasis (Fekete et al. 2021, Barash et al. 2017). Recent studies indicate that Giardia-induced dysbiosis may disrupt microbial fermentation of glycans, leading to altered short-chain fatty acid (SCFA) production and immune modulation (Fekete et al. 2024, Chen et al. 2019).
Prebiotics such as fructooligosaccharides (FOS) are nondigestible carbohydrates that promote the growth of beneficial gut microbes, including Bifidobacterium and Lactobacillus, resulting in increased SCFA production, particularly acetate and propionate (Mahalak et al. 2023). These microbial metabolites act not only as energy sources for colonocytes but also as signaling molecules that regulate intestinal barrier function, mucosal immunity, and systemic metabolic pathways (Iacob et al. 2019). In particular, SCFAs can influence bile acid metabolism via effects on nuclear receptors such as the farnesoid X receptor (FXR), which controls bile acid synthesis through the intestinal FXR–FGF15 (rodents) or FXR–FGF19 (humans) signaling axis (Chen et al. 2019).
The hepatic synthesis of bile acids is a tightly regulated process essential for cholesterol homeostasis, lipid digestion, and host–microbiota signaling (Ciaula et al. 2017). Bile acids are synthesized from cholesterol, mainly coordinated by cholesterol 7α-hydroxylase, encoded by Cyp7a1, the rate-limiting enzyme in bile acid biosynthesis, and the sterol 12α-hydroxylase, encoded by Cyp8b1, both of which are regulated by the nuclear receptor FXR (Chambers et al. 2019). Alterations in the expression of these genes due to microbial, nutritional, or inflammatory factors can shift bile acid synthesis and composition, with downstream effects on metabolism, barrier function, and immune responses (Chen et al. 2019).
This study aimed to investigate the effects of Giardia lamblia infection and FOS supplementation on gut fermentation products, disaccharidase activity, inflammatory markers, and hepatic bile acid synthesis in a gerbil model. By integrating analyses of cecal SCFA profiles, enzyme activities, cytokine levels, and expression of key hepatic genes (Cyp7a1 and Cyp8b1), we sought to elucidate the role of microbiota–host interactions in shaping the metabolic and immunological outcomes of giardiasis and FOS preventive treatment.
MATERIALS AND METHODS
Experimental model and group divisions
Twenty male gerbils (Meriones unguiculatus), aged 4–6 weeks, were used in the experiments. Before the study, all animals were treated with 1% injectable ivermectin to eliminate potential parasitic infections. The absence of enteroparasites was confirmed by standard microscopic examination of fecal samples, conducted every two days before G. lamblia infection, which targeted the detection of cysts, trophozoites, and helminth eggs. The animals were housed under standard laboratory conditions, including a 12-h light/12-h dark cycle and a controlled temperature of 23 ± 3°C, with access to a conventional diet (Nuvilab®) ad libitum. All experimental procedures were conducted according to the protocols approved by the Ethics Committee on Animal Use of Federal University of Minas Gerais (protocol 371/17).
Animals were assigned to four groups, following the protocol described in our earlier study (Ribeiro et al. 2025): uninfected control (CT), infected control (CTIN), uninfected treated with FOS (FOS), and infected treated with FOS (FOSIN). Commercial fructooligosaccharide (FOSVITA-FOS, Vitafor®) was administered daily by oral gavage for 28 days to the FOS and FOSIN groups (250 mg of FOS suspended in 250 µL of distilled water). On day 14 of the experiment, animals in the CTIN and FOSIN groups were orally infected by gavage with 1 × 10⁶ trophozoites of Giardia lamblia (strain GS/M Clone H7-ATCC 50581) suspended in 0.8 mL of phosphate-buffered saline. Trophozoites were kept in culture in TYI-S-33 medium modified by Keister (1983) and supplemented with bovine serum. To confirm Giardia infection, the cecal contents were diluted in water, stained with Lugol’s solution, and examined under an Axiolab-Carl Zeiss microscope (Oberkochen, Germany) with a magnification of 40×.
On the 14th day post-infection, gerbils were anesthetized with intraperitoneal ketamine (100 mg/kg) and xylazine (12 mg/kg). Blood was collected from the inferior vena cava, followed by cervical dislocation. The blood samples were centrifuged at 12,000 × g for 10 minutes at 4°C, and the serum was separated and stored for further analysis. The liver was removed and cecal contents were collected from the cecum. To assess region-specific intestinal responses, the small intestine was dissected from the pyloric sphincter to the ileocecal junction and measured without stretching. Based on total length, the proximal jejunum (10 cm from the duodenojejunal angle) was collected to measure disaccharidase activity, and ileum (10 cm from the ileocecal junction) was separated for cytokine analysis.
All tissue samples were carefully rinsed with cold phosphate-buffered saline (PBS), dried on filter paper, trimmed, and stored at –80°C until further analysis.
Cecal SCFA detection: gas chromatographic analysis
Cecal SCFA extraction was performed according to the method described by Lu et al. (2016). In this procedure, 150 mg of each fresh cecal sample was weighed and suspended in 1.2 mL of sterile distilled water, then homogenized for approximately 3 minutes. The pH of the suspension was adjusted to 2–3 by adding 5 M HCl, followed by a 10-minute incubation at room temperature with occasional shaking. The mixture was then transferred to a polypropylene tube and centrifuged at ~17,000 g (Neofuge 15R, Heal Force) for 10 minutes at 4°C. This centrifugation step was repeated until the supernatant became clear. Cecal SCFAs were measured using gas chromatography (GC) according to a previously described method (Silva et al. 2023).
The supernatant was injected in an Agilent HP7820A GC system equipped with a flame ionization detector. A high-resolution gas chromatography column (INNOWAX – HP, Agilent) of 15 m × 0.25 mm i.d. coated with 0.25μm film thickness was used. Hydrogen was supplied as the carrier gas at a flow rate of 2 mL/min. The initial oven temperature was 100°C, increasing 7 °C/min up to 240°C. The injector split operated at 1/30 at 250°C and the detector temperature was 260°C. The injected sample volume was 1 μL. Peak identification was performed by comparing the retention times of the samples to those of a fatty acid standard analyzed under the same chromatographic conditions.
Measurement of intestinal disaccharidase activity
The proximal jejunum was processed as described by Pereira et al. (2011). The tissue was mechanically homogenized at 300 rpm for 1 minute in an ice bath with 500 µL of PBS. The resulting extract was centrifuged at 5000 g for 10 min at 4°C. The supernatant was used for the measurement of maltase, and lactase activities, as described by Sadek et al. (1986). Protein content was determined by a commercial BCA assay kit (Pierce™ BCA Protein Assay Kit) from Thermo Fisher Scientific (IL, USA).
The determination of enzyme activity was performed by measuring glucose using an enzymatic colorimetric assay (Labtest kit, Brazil) following the guidelines of the manufacturer’s protocols. The reading was performed at 500 nm using a microplate reader – ThermoFisher Multiskan FC 51119000. One unit of disaccharidase activity (U) was defined as the amount of enzyme that catalyzed the release of 1 μmol of glucose per minute at 37°C. The tests were performed in duplicate, and the result was expressed as units of enzyme per mg of protein.
Cytokine detection by flow cytometry in serum and intestine
A portion of the ileum was processed using a tissue homogenizer (300 rpm for 1 minute in an ice bath with 500 µL of PBS), centrifuged at 5,000 g for 10 min at 4°C and the supernatant was collected.
Serum and intestinal cytokines (IL-12, TNF-α, IFN-γ, MCP-1, IL-6, IL-10) were measured using a Cytometric Bead Array (CBA) kit (BD Biosciences, Catalog number 552364), according to the manufacturer’s instructions. All samples were analyzed in technical triplicate, and triplicates showed low intra-assay variability and highly consistent median fluorescence intensities. Samples were acquired on a FACSVerse flow cytometer (BD Biosciences) and data were processed using FCAP Array multiplex analysis software (Soft Flow, Inc., St. Louis Park, MN, USA).
Gene expression: RNA extraction from tissues and quantification by RT-PCR
Total RNA was extracted from liver and intestine using TRI Reagent® (Sigma-Aldrich) and quantified by spectrophotometry using a NanoVue Plus (GE®). cDNA was synthesized from 2 μg of total RNA using oligo(dT) primers and the High-Capacity RNA-to-cDNA™ Kit with RNase inhibitor (Applied Biosystems™), following the manufacturer’s protocol: incubation at 42°C for 10 minutes, followed by 55 °C for 60 minutes.
Real-time PCR (qRT-PCR) was performed on a 7500 Fast Real-Time System (Applied Biosystems®) using the SYBR Green system to assess the hepatic expression of genes involved in bile acid metabolism (Cyp7a1 and Cyp8b1). The qRT-PCR conditions were: 95°C for 2 minutes (1 cycle), 95°C for 5 seconds, 60°C for 30 seconds (40 cycles) as specified by the manufacturer.
The oligonucleotide primers for the target genes in this study were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), based on gene sequences from the NCBI database for gerbil (Meriones unguiculatus) and mouse (Mus musculus). The GAPDH gene was used as an internal control. Each sample was run in triplicate. Melting curves were generated to confirm the specificity of the PCR products. Fold-change in gene expression was calculated using Ct values and the 2^(-ΔΔCt) method. The nucleotide sequences of the primers used are listed in Table I.
Statistical analysis
Outliers were identified using Grubbs’ test (α = 0.05). Outlier detection resulted in exclusions only in the cytokine datasets (serum and intestinal measurements); no outliers were detected in the remaining analyses. When detected, the corresponding data point was removed. The final sample sizes for each cytokine after outlier exclusion are reported in the figure legends. After outlier removal, normality of residuals was assessed using the Kolmogorov–Smirnov test, and all datasets met the assumptions of normal distribution (p > 0.05). The effects of infection (uninfected control vs. infected control), FOS supplementation (uninfected treated vs. infected treated), and their interaction (FOS × infection) were assessed by two-way analysis of variance (ANOVA). When the two-way ANOVA indicated significant main effects or interactions, pairwise differences were evaluated using Tukey’s multiple comparisons post hoc test at p < 0.05. Data are presented as means ± SD. Statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software, USA).
RESULTS
FOS treatment changes SCFA excretion
As shown in Table II, both infection and FOS supplementation increased acetic acid levels, whereas both factors independently reduced butyric acid levels, indicating additive effects. Propionic acid and other SCFAs were significantly increased by FOS supplementation, with no effect of infection. Post hoc comparisons confirmed that these FOS-induced increases occurred in both uninfected and infected animals.
FOS supplementation reduces intestinal maltase activity regardless of infection status
Figure 1 shows intestinal disaccharidase activities. Maltase activity (Fig. 1a) was significantly reduced by FOS supplementation [F(1,16) = 14.30, p = 0.0016], whereas neither infection [F(1,16) = 0.6277, p = 0.4398] nor the interaction [F(1,16) = 0.01418, p = 0.9067] were significant. Post hoc comparisons confirmed reduced maltase activity in FOS-supplemented animals both in uninfected (CT vs. FOS; p = 0.0280) and infected animals (CTIN vs. FOSIN; p = 0.0395), indicating a consistent effect of FOS independent of infection. In contrast, lactase activity (Fig. 1b) was not affected by FOS supplementation or infection, and no interaction was detected [FOS: F(1,16) = 0.1904, p = 0.6684; infection: F(1,16) = 0.6504, p = 0.4318; interaction: F(1,16) = 3.47×10-5, p = 0.9954].
Intestinal disaccharidase activities. a) Maltase activity was significantly reduced by FOS supplementation, while infection had no effect, and no interaction was detected. Different letters indicate significant differences between FOS levels (p < 0.05). Groups without FOS share the letter “a”, and groups supplemented with FOS share the letter “b”. b) Lactase activity was not affected by FOS supplementation or infection, and no interaction was observed. Values are presented as mean ± SD. Two-way ANOVA followed by Tukey’s post hoc test; n = 5.
Effects of FOS supplementation and infection on serum cytokine levels. a) IL-12; b) TNF; c) INF-γ; d) MCP-1; e) IL-10; f) IL-6. Data are presented as mean ± SD. Two-way ANOVA followed by Tukey’s post hoc test was used to assess differences among the four experimental groups (n = 4–5). Different letters indicate significant differences (p < 0.05). Groups sharing the same letter do not differ significantly.
Effects of FOS supplementation and infection on serum cytokine levels
G. lamblia infection significantly increased serum TNF-α levels [F(1,15) = 13.69, p = 0.0021], and this effect was attenuated by FOS supplementation, as supported by a significant interaction between factors [F(1,15) = 15.72, p = 0.0012]. Serum IL-10 also showed a significant interaction [F(1,15) = 9.993, p = 0.0065], with FOS increasing IL-10 in infected mice [F(1,15) = 6.676, p = 0.0200] while producing a different pattern in non-infected animals. No significant effects were detected for serum IL-12, IFN-γ, MCP-1, or IL-6 (p > 0.05).
Effects of FOS supplementation and infection on intestinal cytokine levels
Intestinal IL-10 (Fig. 3e) was significantly affected by both FOS supplementation [F(1,16) = 7.598, p = 0.0147] and infection [F(1,16) = 12.90, p = 0.0027], with no interaction [F(1,16) = 0.5660, p = 0.4635]. Post hoc analysis showed that infection increased IL-10 only in non-supplemented animals (CTIN vs CT; p = 0.0185), while no other pairwise comparisons reached statistical significance. MCP-1 (Fig. 3f) showed a significant FOS × infection interaction [F(1,16) = 6.794, p = 0.0191], but post hoc tests revealed no pairwise differences, indicating a divergent pattern without significant contrasts. Other intestinal cytokines were not significantly affected (p > 0.05).
Effects of FOS supplementation and infection on intestinal cytokine levels. a) IL-12; b) TNF; c) INF-γ; d) MCP-1; e) IL-10; f) IL-6. Data are presented as mean ± SD. Two-way ANOVA followed by Tukey’s post hoc test was used to assess differences among the four experimental groups (n = 4–5). The asterisk indicates a significant effect of infection in non-supplemented animals (CT vs CTIN; p < 0.05).
Technical reproducibility of cytokine measurements
All samples were analyzed in technical triplicate. Although many cytokine concentrations fell below the kit’s limit of detection (LOD), median fluorescence intensities (MFIs) were highly consistent, indicating that the assay produced stable and reproducible signals across gerbil serum and intestinal samples. This consistency supports the reliability of relative comparisons between experimental groups.
Hepatic expression of bile acid–synthesis genes ( Cyp7a1 and Cyp8b1 )
Hepatic Cyp7a1 expression (Fig. 4a) was significantly influenced only by infection [F(1,16) = 5.543, p = 0.0317], with infected animals showing higher expression. Neither FOS [F(1,16) = 1.440, p = 0.2476] nor the interaction [F(1,16) = 0.6994, p = 0.4153] was significant, and post hoc comparisons showed no differences within FOS conditions (p > 0.05).
For Cyp8b1 (Fig. 4b), a significant FOS × infection interaction was detected [F(1,16) = 5.684, p = 0.0298], while neither factor alone was significant (FOS: p = 0.7728; infection: p = 0.1615). Tukey post hoc comparisons revealed no significant differences among the four groups (p > 0.05).
Hepatic expression of Cyp7a1 (a) and Cyp8b1 (b) in control and infected gerbils with or without FOS supplementation. Data are presented as mean ± SD (n = 5). Two-way ANOVA followed by Tukey’s post hoc test. For Cyp7a1, the horizontal line and asterisk above the infected groups (CTIN and FOSIN) indicate the main effect of infection.
DISCUSSION
This study investigated the effects of fructooligosaccharide (FOS) supplementation on intestinal and hepatic responses during experimental giardiasis, focusing on cecal short-chain fatty acid (SCFA) excretion, disaccharidase activity, inflammatory markers, and the expression of genes involved in bile acid metabolism. FOS, a non-digestible carbohydrate with prebiotic properties, is fermented by the gut microbiota, contributing to colonic acidification and the production of SCFAs and other metabolites that promote host health (Dias et al. 2025, Correa et al. 2024, Ashaolu et al. 2021). Acetate, propionate, and butyrate are the primary fermentation products of these prebiotic fibers (Pham et al. 2021).
Short-chain fatty acids (SCFAs) are key metabolites produced by bacterial fermentation of non-digestible carbohydrates and play central roles in intestinal and systemic metabolism (Ashaolu et al. 2021, Pham et al. 2021, Correa et al. 2024). In the present study, both G. lamblia infection and FOS supplementation independently increased fecal acetic acid levels and reduced butyric acid excretion, indicating additive effects of these factors on microbial fermentation.
Alterations in SCFA profiles during giardiasis have been associated with infection-induced dysbiosis and changes in mucin utilization. Fekete et al. (2024) demonstrated that Giardia muris infection in mice induces microbiota disruption (Bhatt et al. 2024) and modifies SCFA production, particularly during the acute phase of infection. Similarly, previous studies have shown that Giardia infection alters gut microbial composition, reduces butyrate-producing bacteria, and shifts fermentation toward acetate and propionate production (Barash et al. 2017, Karpe et al. 2023, Klimczak et al. 2024).
In the present study, propionic acid and other minor SCFAs were significantly increased by FOS supplementation regardless of infection status. In contrast, Mao et al. (2018), studying the effects of 5% FOS supplementation on the fecal microbiota of mice, reported no significant changes in individual SCFA levels, although the relative proportion of butyrate was reduced. These differences highlight the influence of host species, experimental design, dietary composition, and baseline microbiota on metabolic outcomes associated with FOS supplementation.
The reduction in fecal butyrate observed in both infected and FOS-treated animals may reflect altered microbial production and/or increased epithelial utilization. Butyrate is the main energy source for colonocytes and plays a critical role in maintaining epithelial integrity and metabolic homeostasis (Maslowski 2019, Byndloss et al. 2017). Other SCFAs, such as propionate and acetate, serve as energy substrates for the liver and peripheral tissues, respectively (Csernus & Czeglédi 2020). In addition, SCFAs modulate epithelial metabolism, increase luminal acidity, and limit colonization by pathogenic microorganisms (Iacob et al. 2019). Thus, enhanced epithelial uptake and utilization of SCFAs, particularly butyrate, may contribute to the reduced fecal levels observed in FOS-treated infected animals.
Analysis of disaccharidase activities revealed that FOS supplementation significantly reduced maltase activity independently of infection, whereas lactase activity remained unchanged. These findings indicate a selective effect of FOS on carbohydrate digestion rather than a generalized impairment of brush-border function, consistent with our previous observation of preserved intestinal architecture in FOS-treated infected animals (Ribeiro et al. 2025). Previous studies indicate that the effects of FOS on disaccharidase activity vary according to dose, duration, host species, and gut microbiota composition (Csernus & Czeglédi 2020). In the present model, FOS-induced shifts in microbial fermentation and luminal SCFA profiles may modulate maltase expression or activity without affecting lactase, suggesting an adaptive reprogramming of intestinal carbohydrate metabolism.
To assess immune responses, we evaluated a panel of systemic and intestinal cytokines. Infected animals exhibited increased serum TNF-α levels, consistent with reports that Giardia activates innate immune pathways and induces pro-inflammatory cytokine release (Pu et al. 2021). In contrast, FOS treatment significantly increased both serum and intestinal IL-10 levels in infected animals, indicating a shift toward a regulatory immune profile. SCFAs derived from prebiotic fermentation are known to suppress NF-κB signaling and promote regulatory T-cell differentiation, leading to enhanced IL-10 production and resolution of inflammation (Iacob et al. 2019, Mao et al. 2018). These findings support a role for FOS in promoting immune regulation, particularly under inflammatory conditions.
Although MCP-1 exhibited a significant interaction effect in intestinal samples, the absence of significant post hoc differences suggests a divergent response pattern rather than discrete group-specific changes, underscoring the need for cautious interpretation of interaction terms when pairwise contrasts are not detected.
Following the analysis of inflammatory responses, we examined the hepatic expression of genes involved in bile acid metabolism, focusing on Cyp7a1 and Cyp8b1. Bile acids constitute a critical link between microbial activity and host metabolic regulation. In this study, G. lamblia infection significantly increased hepatic Cyp7a1 expression, independent of FOS supplementation, suggesting enhanced bile acid synthesis during infection. Cyp7a1 is the rate-limiting enzyme of the classical bile acid synthesis pathway (Chiang 2017, Ciaula et al. 2017). Cyp8b1 expression exhibited a significant interaction between infection and FOS supplementation, although no post hoc differences were detected among individual groups. As Cyp8b1 regulates the balance between cholic and chenodeoxycholic acids, such interaction effects may reflect subtle shifts in bile acid regulation during combined dietary and infectious challenges (Chambers et al. 2019). Similar alterations in bile acid signaling and FXR activity have been described in murine models of enteric infection and dysbiosis (Lane et al. 2023, Klimczak et al. 2024).
Although direct measurements of intestinal FXR–FGF15 signaling and bile acid composition were not performed, existing evidence supports a link between infection-induced dysbiosis, altered bile acid pools, and hepatic regulation of bile acid synthesis genes (Chen et al. 2019, Riba et al. 2020).
Overall, our findings indicate that FOS primarily modulates intestinal fermentation patterns, maltase activity, and immune regulation, whereas Giardia infection is a major driver of systemic inflammation and hepatic bile acid gene expression. The interaction between FOS supplementation and infection was most evident in immune parameters, highlighting the context-dependent effects of dietary prebiotics. Further studies integrating microbiota profiling, bile acid composition, and intestinal signaling pathways will be essential to clarify the mechanisms underlying these observations.
Limitations
This study has several limitations that should be considered when interpreting the results. First, the relatively small sample size (n = 5 per group) may limit statistical power to detect subtle differences and increase the risk of Type II errors. While consistent trends were observed, a larger cohort would strengthen the reliability and generalizability of the findings.
Second, we did not evaluate intestinal FXR or its downstream target FGF15, which would help confirm the proposed mechanism linking secondary bile acids to reduced FXR activation and provide evidence for gut–liver feedback regulation of bile acid synthesis.
Another key limitation is the absence of microbiota profiling. Since FOS is known to modulate gut microbial composition and functions, including species involved in bile acid deconjugation and 7α-dehydroxylation, microbiome analyses would be essential to establish causal links between FOS supplementation, microbial shifts, and altered bile acid metabolism. Additionally, bile acid composition and total bile flow were not assessed, limiting correlation of hepatic gene expression (e.g., Cyp7a1, Cyp8b1) with functional outcomes.
Finally, cytokine levels were measured using the Mouse Inflammation CBA Kit, which has not been formally validated for use in gerbils and was employed due to the lack of species-specific reagents. Absolute cytokine concentrations should therefore be interpreted with caution, especially for analytes near or below the manufacturer’s reported limits of detection for mouse cytokines. Nevertheless, the assays produced consistent and reproducible signals across technical replicates, and the observed cytokine patterns were biologically coherent across experimental groups. Thus, while absolute quantification may be limited, the data remain informative for relative comparisons and for identifying trends in inflammatory responses associated with Giardia infection and FOS supplementation.
Addressing these limitations in future studies will help to clarify the complex interactions among dietary prebiotics, gut microbiota, bile acid signaling, and host immune responses in giardiasis.
CONCLUSIONS
Our findings show that Giardia lamblia infection alters gut microbial fermentation and immune responses, as reflected by changes in fecal short-chain fatty acids and increased serum TNF-α levels. Infection was also associated with increased hepatic Cyp7a1 expression, indicating that giardiasis can influence pathways related to bile acid synthesis.
FOS supplementation mainly affected intestinal and immune parameters. Regardless of infection, FOS modified SCFA profiles, increasing propionate and other minor SCFAs, and consistently reduced intestinal maltase activity without affecting lactase, suggesting changes in carbohydrate metabolism linked to microbial fermentation. In infected animals, FOS promoted a more regulatory immune profile, characterized by increased IL-10 levels in serum and intestinal tissue.
Overall, these results highlight the interaction between parasitic infection, diet, microbial metabolism, and host immune regulation. While this study provides evidence that prebiotic supplementation can modulate inflammatory and metabolic responses during giardiasis, further studies are needed to better define the underlying mechanisms. Future work should include analyses of microbial composition, bile acid profiles, and gut–liver signaling pathways. Incorporating microbiome sequencing approaches, such as 16S rRNA gene profiling or metagenomics, would be essential to directly link observed changes in SCFA profiles and bile acid outcomes to specific bacterial taxa and functional genes (e.g., bile salt hydrolases), providing stronger mechanistic support for the microbiota-driven effects suggested by the present findings.
Acknowledgements
We would like to express our deepest gratitude to Lorena Junia de Souza Santos, Cytometry Platform Technician at the René Rachou Institute - Belo Horizonte, MG, for her invaluable expertise and guidance. Her generous assistance with cytokine analysis by flow cytometry in both serum and intestinal samples greatly enhanced the quality and depth of this study. Her brilliance, patience, and dedication were truly inspiring, and we are sincerely thankful for her support. This work was supported by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, grant number APQ 03545-18).
References
-
ALLAIN T, FEKETE E, SOSNOWSKI O, DESMONTS DE LAMACHE D, MOTTA JP, LEGER D, FEENER T, REIMER RA & BURET AG. 2021. High-fat diet increases the severity of Giardia infection in association with low-grade inflammation and gut microbiota dysbiosis. Sci Rep 11(1): 18842. doi: 10.1038/s41598-021-98262-8.
» https://doi.org/10.1038/s41598-021-98262-8 -
ASHAOLU TJ, ASHAOLU JO & ADEYEYE SAO. 2021. Fermentation of prebiotics by human colonic microbiota in vitro and short-chain fatty acids production: a critical review J Appl Microbiol 130(3): 677-687. doi: 10.1111/jam.14843.
» https://doi.org/10.1111/jam.14843 -
BARASH NR, MALONEY JG, SINGER SM & DAWSON SC. 2017. Giardia alters commensal microbial diversity throughout the murine gut. Infect Immun 85(6): e00948-16. doi: 10.1128/IAI.00948-16.
» https://doi.org/10.1128/IAI.00948-16 -
BHATT AP ET AL. 2024. Giardia antagonizes beneficial functions of indigenous and therapeutic intestinal bacteria during protein deficiency. Gut Microbes 16(1): 2421623. doi: 10.1080/19490976.2024.2421623.
» https://doi.org/10.1080/19490976.2024.2421623 -
BYNDLOSS MX ET AL. 2017. Microbiota-activated PPAR-γ-signaling inhibits dysbiotic Enterobacteriaceae expansion. Science 357(6351): 570-575. doi:10.1126/science.aam9949.
» https://doi.org/10.1126/science.aam9949 -
CHAMBERS KF, DAY PE, ABOUFARRAG HT & KROON PA. 2019. Polyphenol effects on cholesterol metabolism via bile acid biosynthesis, CYP7A1: a review. Nutrients 11(11): 2588. doi: 10.3390/nu11112588.
» https://doi.org/10.3390/nu11112588 -
CHEN ML, TAKEDA K & SUNDRUD MS. 2019. Emerging roles of bile acids in mucosal immunity and inflammation. Mucosal Immunol 12(4): 851-861. doi: 10.1038/s41385-019-0162-4.
» https://doi.org/10.1038/s41385-019-0162-4 -
CHIANG JYL. 2017. Recent advances in understanding bile acid homeostasis. F1000Res 6: 2029. doi: 10.12688/f1000research.12449.1.
» https://doi.org/10.12688/f1000research.12449.1 -
CIAULA A, GARRUTI G, BACCETTO RL, MOLINA-MOLINA E, BONFRATE L, WANG DQH & PORTINCASA P. 2017. Bile acid physiology. Ann Hepatol 16 (Suppl. 1: s3-105): s4-s14. doi: 10.5604/01.3001.0010.5493.
» https://doi.org/10.5604/01.3001.0010.5493 -
CORREA AC, LOPES MS, PERNA RF & SILVA EK. 2024. Fructan-type prebiotic dietary fibers: Clinical studies reporting health impacts and recent advances in their technological application in bakery, dairy, meat products and beverages. Carbohydr Polym 323: 121396. doi: 10.1016/j.carbpol.2023.121396.
» https://doi.org/10.1016/j.carbpol.2023.121396 -
CSERNUS B & CZEGLÉDI L. 2020. Physiological, antimicrobial, intestine morphological, and immunological effects of fructooligosaccharides in pigs. Arch Anim Breed 63: 325-335. https://doi.org/10.5194/aab-63-325-2020.
» https://doi.org/10.5194/aab-63-325-2020 -
DIAS GS ET AL. 2025. Fructooligosaccharides: A comprehensive review on their microbial source, functional benefits, production technology, and market prospects. Processes 13(4): 1252. https://doi.org/10.3390/pr13041252.
» https://doi.org/10.3390/pr13041252 -
FEKETE E, ALLAIN T, SIDDIQ A, SOSNOWSKI O & BURET AG. 2021. Giardia spp. and the gut microbiota: dangerous liaisons. Front Microbiol 11: 618106. doi: 10.3389/fmicb.2020.618106.
» https://doi.org/10.3389/fmicb.2020.618106 -
FEKETE E, ALLAIN T, SOSNOWSKI O, ANDERSON S, LEWIS IA & BURET AG. 2024. Giardia spp.-induced microbiota dysbiosis disrupts intestinal mucin glycosylation. Gut Microbes 16(1): 2412676. doi: 10.1080/19490976.2024.2412676.
» https://doi.org/10.1080/19490976.2024.2412676 -
IACOB S, IACOB DG & LUMINOS LM. 2019. Intestinal microbiota as a host defense mechanism to infectious threats. Front Microbiol 9: 3328. doi: 10.3389/fmicb.2018.03328.
» https://doi.org/10.3389/fmicb.2018.03328 -
KARPE AV ET AL. 2023. Gut microbial perturbation and host response induce redox pathway upregulation along the gut-liver axis during giardiasis in C57BL/6J mouse model. Int J Mol Sci 24(2): 1636. doi: 10.3390/ijms24021636.
» https://doi.org/10.3390/ijms24021636 -
KEISTER DB. 1983. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77(4): 487-488. doi: 10.1016/0035-9203(83)90120-7.
» https://doi.org/10.1016/0035-9203(83)90120-7 -
KLIMCZAK S, PACKI K, RUDEK A, WENCLEWSKA S, KUROWSKI M, KURCZABIŃSKA D & ŚLIWIŃSKA A. 2024. The influence of the protozoan Giardia lamblia on the modulation of the immune system and alterations in host glucose and lipid metabolism. Int J Mol Sci 25(16): 8627. doi: 10.3390/ijms25168627.
» https://doi.org/10.3390/ijms25168627 -
LANE JM, BROSSCHOT TP, GATTI DM, COU GAUTHIER CM, LAWRENCE KM, PLUZHNIKOVA V & REYNOLDS LA. 2023. Chronic small intestinal helminth infection perturbs bile acid homeostasis and disrupts bile acid signaling in the murine small intestine. Front Parasitol 2: 1214136. doi: 10.3389/fpara.2023.1214136.
» https://doi.org/10.3389/fpara.2023.1214136 -
LU Y, FAN C, LI P, LU Y, CHANG X & QI K. 2016. Short chain fatty acids prevent high-fat-diet-induced obesity in mice by regulating G protein-coupled receptors and gut microbiota. Sci Rep 6: 37589. doi: 10.1038/srep37589.
» https://doi.org/10.1038/srep37589 -
MAHALAK KK, FIRRMAN J, NARROWE AB, HU W, JONES SM, BITTINGER K, MOUSTAFA AM & LIU L. 2023. Fructooligosaccharides (FOS) differentially modifies the in vitro gut microbiota in an age-dependent manner. Front Nutr 9: 1058910. doi: 10.3389/fnut.2022.1058910.
» https://doi.org/10.3389/fnut.2022.1058910 -
MAO B, GU J, LI D, CUI S, ZHAO J, ZHANG H & CHEN W. 2018. Effects of different doses of fructooligosaccharides (FOS) on the composition of mice fecal microbiota, especially the Bifidobacterium composition. Nutrients 10(8): 1105. doi: 10.3390/nu10081105.
» https://doi.org/10.3390/nu10081105 -
MASLOWSKI KM. 2019. Metabolism at the centre of the host–microbe relationship. Clin Exp Immunol 197(2): 193-204. doi: 10.1111/cei.13329.
» https://doi.org/10.1111/cei.13329 -
PEREIRA DF, CAZAROLLI LH, LAVADO C, MENGATTO V, FIGUEIREDO MSRB, GUEDES A, PIZZOLATTI MG & SILVA FRMB. 2011. Effects of flavonoids on α-glucosidase activity: potential targets for glucose homeostasis. Nutrition 27(11-12): 1161-1167. doi: 10.1016/j.nut.2011.01.008.
» https://doi.org/10.1016/j.nut.2011.01.008 -
PHAM VT, CALATAYUD M, ROTSAERT C, SEIFERT N, RICHARD N, ABBEELE PVD, MARZORATI M & STEINERT RE. 2021. Antioxidant vitamins and prebiotic FOS and XOS differentially shift microbiota composition and function and improve intestinal epithelial barrier in vitro. Nutrients 13(4): 1125. doi: 10.3390/nu13041125.
» https://doi.org/10.3390/nu13041125 -
PU X ET AL. 2021. Giardia duodenalis induces proinflammatory cytokine production in mouse macrophages via TLR9-Mediated p38 and ERK signaling pathways. Front Cell Dev Biol 9: 694675. doi: 10.3389/fcell.2021.694675.
» https://doi.org/10.3389/fcell.2021.694675 -
RIBA A ET AL. 2020. Disturbed gut microbiota and bile homeostasis in Giardia-infected mice contributes to metabolic dysregulation and growth impairment. Sci Transl Med 12(565): eaay7019. doi: 10.1126/scitranslmed.aay7019.
» https://doi.org/10.1126/scitranslmed.aay7019 -
RIBEIRO MRS, CARVALHO CF, GOMES MA, CASSALI GD & OLIVEIRA DR. 2025. Fructooligosaccharides (FOS) intake modulates oxidative stress and restores gut morphology in Giardia lamblia-infected gerbils. An Acad Bras Cienc 97: e20250346. DOI 10.1590/0001-3765202520250346.
» https://doi.org/10.1590/0001-3765202520250346 -
SADEK MG, BORGES DR & MISZPUTEN SJ. 1986. Intestinal activity of disaccharidases in schistosomiasis mansoni. Study of the course in mice with various degrees of infestation. Rev Inst Med Trop Sao Paulo 28(2): 67-73. doi: 10.1590/s0036-46651986000200001.
» https://doi.org/10.1590/s0036-46651986000200001 -
SILVA NC, SILVA YOB, TAKAHASHI JA, ANDRADE SP, CASSALI GD & OLIVEIRA DR. 2023. Dietary lipids management: are there any benefits from the prevention and treatment of 4T1 murine breast carcinoma? An Acad Bras Cienc 95: e20210330. doi: 10.1590/0001-3765202320210330.
» https://doi.org/10.1590/0001-3765202320210330
Edited by
-
Handling editor
Helton Santiago
The data supporting the findings of this study are available from the corresponding author upon reasonable request.
