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Nano-chitosan hydrogel alleviates Candida albicans-induced health alterations in Nile tilapia (Oreochromis niloticus): antioxidant response, neuro-behaviors, hepato-renal functions, and histopathological investigation

BMC Veterinary Research volume 21, Article number: 159 (2025) Cite this article

Abstract

Background

Candida albicans infection induces economic losses in aquaculture practices. Currently, the success of the nanotechnology field has gained more consideration in the aquaculture sector as it bestows favorable impacts in remedies in comparison to traditional practices.

Objective

The present study was conducted to assess the role of nano chitosan gel (NCG) exposure via water in managing the deteriorating impacts triggered by C. albicans in Nile tilapia, Oreochromis niloticus. Hepato-renal function, behavioral and stress response, neurological function, hepatic antioxidant/oxidant status, and histopathological architectures were investigated.

Methods

A total of 160 fish (average weight: 50.00 ± 6.30 g) were randomly assigned to four groups, each with four replicates: control, NCG, C. albicans, and NCG + C. albicans. The NCG was applied as bath treatment at a concentration of 75 µg/L for ten days.

Results

The outcomes demonstrated that the C. albicans challenged fish exhibited obvious behavioral alterations including loss of equilibrium, surfacing, abnormal swimming and movement, and aggression. Infection with C. albicans caused an elevation in hepato-renal biomarkers (alanine and aspartate aminotransferases, alkaline phosphatase, urea, and creatinine), stress-related indices (glucose, cortisol, nor-epinephrine, and 8-hydroxy-2-deoxyguanosine), and lipid peroxides (malondialdehyde). Moreover, it caused a noticeable decline in the hepatic antioxidant indices (total antioxidant capacity and reduced glutathione content) and acetylcholinesterase activity. The hepatic, renal, and brain architectures were severely damaged by the C. albicans challenge, exhibiting significant fatty changes, necrosis, vacuolation, and congestion. Remarkably, the aqueous application of NCG in the C. albicans-challenged fish ameliorated all the aforementioned biomarkers and facilitated the regeneration of histopathological changes.

Conclusion

Overall, the application of NCG in the aquatic environment is an effective tool for managing C. albicans infection in Nile tilapia. Moreover, it can be utilized in combating stress conditions in the aquaculture sector.

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Introduction

Recently, fungal infections have negatively altered fish health, resulting in many diseases and higher mortalities [1, 2]. Among them, an opportunistic fungal pathogen called Candida albicans typically lives in the mucosal areas and digestive tracts of birds and mammals [3]. C. albicans is sometimes isolated from aquatic environments that receive municipal wastewater. It serves as a sign of recent fecal contamination [4]. It can infect various fish species inducing skin ulcerations, and hemorrhages, and in turn, elevating the chance for secondary bacterial infections [5,6,7]. It is a pathogenic fungus known to be one of the causative agents of higher fish mortality potentiating economic losses [8]. Particularly, in Nile tilapia (Oreochromis niloticus), it severely destroys epithelial tissues of gills, inducing mortality because of respiratory failure [9]. This probably highlights the fact that C. albicans generate proteases, hemolysins, and esterase—components that contribute to its virulence [10, 11]. Also, It could disseminate in the larva of zebrafish (Danio rerio) through hindbrain route infection and proliferate inducing losses [12]. Moreover, It can pass through blood blood-brain barrier of mammals causing meningitis [13], however, no study focused on the neurotoxicity pathways of C. albicans in Nile tilapia.

Chitosan, a polymer of natural origin (chitin), possesses numerous advantageous properties such as being non-toxic, biocompatible, and biodegradable [14]. It has been frequently utilized to overcome fungal pathogens due to its features [15], particularly against drug-resistant C. albican. This is achieved through binding the higher molecular-weight chitosan with the cell membrane and biofilm, inducing obstruction of nutrient transport [16,17,18]. Lately, a novel approach has emerged, utilizing natural products or nanoparticles (NPs) loaded with them in fish diets or water, to minimize the rate of infections and sustain fish performance [19, 20]. Currently, developing NPs in aquaculture practices has been verified as more successful in boosting immune function and as antimicrobials [21,22,23,24]. Chitosan NPs are effectively utilized in fish because of their safety and effectiveness in provoking growth, and immune-antioxidant response, besides defeating microbial growth in farmed fish [25,26,27]. They have been used efficiently with Ocimum basilicum to boost the health of Nile tilapia [28]. Moreover, generating chitosan-derived NPs can boost the antifungal activity of chitosan even more. This was most likely brought about by the greater cellular absorption, larger surface area, nanosize, and higher surface-to-charge density [29]. In this regard, prior report exhibited their potent antifungal activity against C. albicans [30].

In a recent time, nano-gels (NGs) have been utilized as transporters for bioactive particles, drugs, proteins, and peptides, supplying them to the goal sites that can upkeep the revival of injured tissues [31]. The effectiveness of NGs as a vector is dominated by numerous features, including higher permeability [32], improved bioavailability [33], stability, power to pierce the small capillaries, and powerful drug release [34]. In addition, the presence of a 3D network of conjugated polymers allows precise distribution to the target site and treatment of diseases [35]. Nano chitosan gel (NCG) has been proven brilliant efficacy against (≥ 90% inhibition) biofilms of C. albicans [36]. In zebrafish, NCG did not trigger acute embryotoxicity up to 100 µg/mL [37]. Despite these benefits, its application in aquaculture is still scarce.

Nile tilapia is a universally cultivated fish species that is very prevalent among fish farmers. It is distinguished by a speedy rate of growth and higher palatability [38]. In advance, aqueous NCG has a promising role in enhancing blood indices, immune function, immune-associated genes, and disease resistance of Nile tilapia [39]. No studies are addressing the use of NCG in boosting the tissue antioxidants and architectures as well as its impact on behavior and neuro-stress-related biomarkers against fungal infection in fish. Subsequently, the present work is a pioneering step to investigate the promising role of NCG in Nile tilapia to alleviate the stress, hepato-renal, neuro-ethological, and histopathological alterations induced by C. albicans.

Materials and methods

NCG synthesis and characterization

For the synthesis of NCG, two steps have been carried out; the primary one is the separation of chitosan NPs as defined by Ismail et al. [40]. The other step is using chitosan NPs with a hybrid carbopol gel to produce NCG. Concisely, 50 mg chitosan was used to dissolve in 100 mL acetic acid and mixed for 120 min at 60 °C till obtaining a clear solution. Then, 20 mL of sodium tripolyphosphate was added until a white colloid was obtained. The other product of the synthesis protocol was washed out using a centrifuge at 20,000 rpm, where 20 mg of chitosan NPs was added to 20 mg carbopol, liquefied in 50 mL of deionized distilled water, and agitated to obtain a homogenous solution. Then, a drop-by-drop of trimethylamine (20%) was added until a thick gel was shaped. The characterization measures were separated into three classes: morphology, index, and identification based on a previous study [41].

Ethical approval and acclimation of fish

The experimental measures were accredited by Zagazig University’s Animal Research Ethics Committee, Egypt (ZUIACUC-2-F-333-2022), and followed the NIH guidelines for experimental animal care. Fish (average body weight: 50.00 ± 6.30 g) were collected from Al-Abbassa Fish Hatchery in the Sharkia Governorate, Egypt, and transported in plastic bags. Upon arrival, they were maintained for ten days in 70 L aquaria (80 × 40 × 30 cm) for acclimatization. Compressed air was pumped into each aquarium using air stones for well aeration. Fish did not have any history of disease outbreaks and did not reveal any clinical abnormalities. Prior to the experiment, CCAC [42] guidelines were employed to perform a standard check of the fish’s health. A daily partial water exchange (25%) was carried out and the fish were offered a basal diet (crude protein: 36.46%; Fat: 11.09%; crude fiber: 3.28%) at the rate of 3% of their body weight twice daily (8:00 and 4.00). A daily physicochemical assessment of the rearing water was performed throughout the acclimation and experimental period by taking water specimens from each aquarium. For monitoring water quality metrics [43], involving water temperature, dissolved oxygen, pH, and unionized ammonia, the portable oxygen meter (Jenway, London, UK), digital mini-pH meter (Fisher Scientific, Denver, USA), and multi-parameters ion Analyzer (HANNA Instruments, Rhode Island, USA) were employed, respectively. The recorded measurements were as follows: 25.00 ± 2.00 °C for temperature, 7.00 ± 0.1 for pH, 6.00 ± 0.40 mg/L for dissolved oxygen, and 0.01 ± 0.07 mg/L for unionized ammonia. These variables were within the acceptable limits for aquaculture [44].

Fungal strain (C. albicans)

The pathogenic C. albicans strain was isolated from naturally infected Nile tilapia in the Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Egypt, and proved to be virulent. This isolate was recognized using standard biochemical traits and the VITEK® 2 system (Bio-M’erieux, Marcy l’Etoile, France). C. albicans was exposed to cultivation on sabaroud dextrose agar (Hi-media, India) and then incubated for 48 h at 24 °C [7]. The colonies were chosen and immersed in sterile phosphate buffer saline (PBS). The lethal dose (LD50) of C. albicans was assessed and recorded at 6.4 × 108 CFU/mL for C. albicans [39]. A dose of 1.8 × 107 CFU/mL was designated for the experiment.

Experimental protocol

Prior to any treatment, fish were first anesthetized by being exposed to 100 mg/L of benzocaine solution to facilitate handling and decrease any stress exposure [45]. Fish (n = 160) were randomly chosen and divided into four groups in quadruplicate (40 fish/ group; 10 fish/ replicate) for ten days and each replicate was placed in a 70 L aquarium with adequate airflow. The first (control) group was not exposed to NCG or infected with C. albicans. The second (NCG) group was exposed to NCG and was not infected with C. albicans. The third (C. albicans) group was not exposed to NCG, but it was infected with C. albicans. The fourth (NCG + C. albicans) group was exposed to NCG and infected with C. albicans. The infection with C. albicans was applied by intraperitoneal inoculation of fish with 0.2 mL (1.8 × 107 CFU/mL) of C. albicans fungal suspension [7]. Moreover, the NCG solution was added at a dose of 75 µg/L to the aquarium water [39] after the appearance of clinical symptoms of C. albicans infection (lack of body reflexes, irregular swimming along with skin darkening, and some hemorrhages). To get rid of the excretory wastes, siphoning was done daily. After the full water change (three times per week), a freshly made NCG solution was introduced to keep their levels stable.

Behavioral assessment and sampling

Tracking of behavioral observations was done throughout 11 min for each aquarium twice a day, in the morning (8:00–10:00 am) and afternoon (1:00–3:00 pm) throughout the trial period [46]. The behavior of fish in each aquarium was recorded using the instantaneous sampling method through a direct visualization procedure by using a stopwatch and digital timer (NTT08). Each behavior was observed at 60-second intervals by one observer. The frequencies of behavior were recorded to calculate the number of fish that engaged to perform each behavior per unit of time (m-1). Fish behavioral patterns included loss of equilibrium [47], surfacing [48], swimming [49], resting [50], abnormal movement [51], and aggressive [52].

At the end of the study, fish were carefully chosen (9 fish/group) and anesthetized by immersing in benzocaine (100 mg/L) [45]. Then, blood was collected from the caudal blood vessels using sterile syringes without the addition of anticoagulant and reserved for 6 h at room temperature. Blood was exposed to centrifugation at 1750 ×g for 10 min, and serum samples were utilized for conducting biochemical assays. Moreover, the brain, liver, and kidney tissues of nine fish per group were used as samples for the neurological and antioxidant/oxidant assays as well as histopathological investigations.

Hepato-renal functions assay

Application of biochemical assessments for the activity of serum alanine and aspartate aminotransferases (ALT& AST) and alkaline phosphatase (ALP) enzymes as well as the levels of urea and creatinine using commercial kits of Biodiagnostic Co., Egypt (Cat. No.; AL 1031, AS 1061, AP 1020, UR 2110, CR 1250), respectively [53,54,55,56].

Stress-related assays

Following the protocol established by Teixeira et al. [57], blood glucose (GLU) levels were measured using the glucose oxidase/glucose peroxidase reaction Cortisol hormone and nor-epinephrine (NOR) were distinguished in fish serum by ELISA commercial kit (My-Biosource Inc., San Diego, California, USA) under Cat. No. of MBS704055 and MBS025809, respectively.

Neuro-related assays

Spectrophotometric measurements were made using brain tissue samples to determine the values of 8-hydroxy-2-deoxyguanosine (8-OHdG) and neurotransmitter (acetylcholine, AchE). The brain homogenates were subjected to centrifugation for 15 min at 3000 rpm at 5 °C after using 15 mL of 150 mM NaCl for homogenization. The value of 8-OHdG was estimated using a commercial ELISA kit (MyBiosource Inc., San Diego, California, USA) with a Cat. No. MBS1601729 following manufacturer’s guidance. Firstly, all standards, reagents, and samples were prepared. The ELISA reagent and samples were added into each well and incubated for 1 h at 37 °C. After that, the plates were washed five times. Then, substrate solutions A and B were added and incubation occurred for 10 min at 37 °C. Finally, a stop solution was administered, the color was developed, and the optical density was determined within 10 min at 450 nm.

Furthermore, the level of AchE was estimated at 450 nm detection wavelength based on the protocol illustrated in the report of Ellman et al. [58] using a commercial kit (My-Biosource Inc., San Diego, California, USA) under Cat. No. MBS280290.

Hepatic antioxidant/oxidant assays

The hepatic tissues were splashed three times using a cold NaCl solution (0.9%) prior to homogenization in PBS (pH 7.5). Afterward, the homogenates were subjected to a 15-minute cold centrifugation at 3000 rpm. The supernatants were cautiously gathered in a sterile tube to assess antioxidant/oxidant indicators based on the Fernandez-Botran et al. [59] approach. These indicators were assayed using commercial kits (Biodiagnostic Co., Cairo, Egypt) based on the manufacturer’s directions.

The colorimetric approach was used to evaluate the total antioxidant capacity (TAC; Cat. NO. TA 25 13) following Koracevic et al. [60] method. The reaction of antioxidants in the sample with a specified quantity of externally given hydrogen peroxide (H2O2), which results in the removal of a defined amount of H2O2, is used to quantify the anti-oxidative capacity. The conversion of 3, 5, dichloro-2-hydroxyl benzene-sulphonate to a colorful product in an enzymatic reaction provides a colorimetric measurement of the remaining H2O2. At first, H2O2 as a substrate was diluted and then, a working reagent was formed from the equivalent amount of enzyme buffer and chromogen. About 0.5 mL of sample and blank was thoroughly mixed with H2O2 and incubated at 37 °C for 10 min. Next, the working reagent was added to both, thoroughly mixed, and incubated for 5 min at 37 °C. The TAC was computed after the absorbance of the blank and sample were promptly measured at 505 nm.

To determine the amount of reduced glutathione (GSH; Cat. NO. GR 25 11) colorimetrically, the approach of Beutler et al. [61] was followed. It is based on the conversion of, 5′-dithiobis 2-nitrobenzoic acid into a stable yellow-colored compound (5-mercapto2-nitrobenzoic acid) by GSH. A mixture of 1.8 mL of distilled water, 0.2 mL of sample homogenate, 3 mL of the precipitating agent, and sulfosalicylic acid was formed. A 3000 rpm centrifugation was performed on this mixture for 4 min. After that, the supernatant was combined with 4.5 mL of Ellman’s reagent in a 0.5 mL amount. Then, 4 mL of phosphate buffer, 0.5 mL of the diluted precipitating agent, and 0.5 mL of Ellman’s reagent were coupled. The reaction mixture’s absorbance at 412 nm was measured within 30 min of color formation.

Furthermore, malondialdehyde (MDA; Cat. NO. MD 25 29) was calculated by thiobarbituric acid approach [62]. The basis of this assay is the formation of a pink thiobarbituric acid reactive substance (colored final product) by the reaction of MDA with thiobarbituric acid in an acidic solution at 95 °C for 30 min. The absorbance of this final product was at 534 nm.

Histopathological investigation

Liver, kidney, and brain tissues were collected and fixed in neutral buffered formalin 10% for 24 h. Specimens were dehydrated in graded ethanol (70%, 80%, 90 -100%) and cleared in xylene I followed by xylene II, then embedded in melted paraffin wax I followed by melted paraffin II. We obtained about 5 μm paraffin sections by using automated microtome then stained with Hematoxylin and Eosin (H & E) and examined microscopically for any histological alterations [63]. All section photos were taken using a Swift microscope associated with a Swift digital camera. The histopathological scoring was estimated by semiquantitative methods as follows: “0 = no, 1 = mild, 2 = moderate, and 3 = severe alterations” [64].

Data analysis

Prior to the statistical assay, the normality of the data was checked by using the Shapiro-Wilk normality test. Following that, a one-way analysis of variance (ANOVA) was employed to analyze the results using SPSS ver. 21 (IBM Corp., Armonk, USA). The Duncan test was implemented as a post-hoc analysis to check whether the means varied at a P-value of less than 0.05. The data were shown as mean ± standard error (SE).

Results

NCG characterization

The morphology of NCG appeared as spherical nanoparticles of 60–70 nm size as depicted by transmission electron microscopy (TEM, Fig. 1A) and 3D atomic force microscopy (AFM; Fig. 1B). High purity NCG’s amorphous nature was demonstrated via X-ray diffraction (XRD; Fig. 1C). The measured zeta potential of + 7 mV suggested a moderately positive surface charge on NCG (Fig. 1D). Moreover, a hydrodynamic diameter of approximately 68 nm of NCG was resulted by dynamic light scattering (DLS; Fig. 1E).

Fig. 1
figure 1

Characterization images of nano chitosan gel. A TEM (100 nm). B 3D AFM. C XRD. D Zeta potential. E DLS

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Impact on behavioral observation

The results demonstrated in Table 1 revealed that the C. albicans infection significantly (P < 0.001) altered the behavioral patterns. Fish of the C. albicans group showed significant (P < 0.001) high frequencies of loss of equilibrium, surfacing, abnormal swimming, and resting as well as abnormal movement (vertical and circular). Moreover, increasing the frequencies of most items of aggressive behavior as chasing, mouth pushing, approach, and fleeing induced by C. albicans infection. The fin tugging and butting did not alter significantly. On the other hand, using NCG as a treatment for C. albicans infection (NCG + C. albicans) significantly (P < 0.001) decreased these behavioral alterations relative to the non-treated group (C. albicans). There were no discernible differences were noted between NCG and control groups concerning behavioral traits.

Table 1 Impact of nano chitosan gel (NCG) bath and/or C. albicans infection on frequencies of behavioral pattern (m−1( of Nile tilapia for ten days
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Impact on hepato-renal function variables

Hepato-renal function levels (ALT, AST, ALP, urea, and creatinine) fluctuated in an indiscernible manner between the control and NCG groups (Table 2). C. albicans infection resulted in a noticeable increase (P < 0.001) in these variables as matched to the control. Treatment of the infected group with NCG (NCG + C. albicans) declined these biomarkers compared to the non-treated one (C. albicans).

Table 2 Impact of nano chitosan gel (NCG) bath on hepato-renal variables of Nile tilapia experimentally infected with C. albicans for ten days
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Impact on stress and neuro-related variables

Table 3 reveals non-observable alterations in the value of stress-related variables (GLU, cortisol, and NOR) and neuro-related variables (8-OHdG and AchE) between control and NCG groups. A dramatic rise (P < 0.001) in GLU, cortisol, NOR, and 8-OHdG levels and a decline (P < 0.001) in the AchE value of the C. albicans group with respect to the control. On the contrary, these variables significantly declined (P < 0.001) in the treated group (NCG + C. albicans) compared with the C. albicans group except for AchE activity that showed marked elevation (P < 0.001).

Table 3 Impact of nano chitosan gel (NCG) bath on stress and neuro-related variables of Nile tilapia experimentally infected with C. albicans for ten days
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Impact on antioxidant/oxidant variables

Figure 2 (A–C) demonstrates a noteworthy rise (P < 0.001) in TAC and GSH levels and no significant change in the MDA level of the NCG group as matched to the control. C. albicans infection caused a considerable decline (P < 0.001) in the TAC and GSH and an increase in the MDA value relative to the control. Treatment of C. albicans with NCG improved these metrics relative to the non-treated group.

Fig. 2
figure 2

Impact of nano chitosan gel (NCG) bath on hepatic antioxidant/oxidant variable of Nile tilapia experimentally infected with C. albicans for ten days. A Total antioxidant capacity (TAC; P < 0.001). B Reduced glutathione content (GSH; P < 0.001). C Malondialdehyde (MDA; P < 0.001). Bars (means ± SE) that do not share similar superscripts differ substantially (One way ANOVA; P < 0.05)

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Histopathological outcomes

Normal histo-architectures were observed in the hepato-pancreatic tissues of the control and NCG groups including typical hepatic cords, pancreatic acini, sinusoids, and portal veins (Fig. 3A & B). While markedly distributed fatty changes represented by clear intra-cytoplasmic vacuole with peripherally located nuclei were obvious in the hepato-pancreas of the C. albicans group. As well, necrotic pancreatic acini with pyknotic nuclei were observed (Fig. 3C). These changes were declined in the NCG + C. albicans group, where less number of fatty degenerated cells and amelioration in pancreatic acinar epithelium was seen (Fig. 3D).

Fig. 3
figure 3

Photomicrographs of hepato-pancreas sections (H&E). A & B Hepato-pancreases of the control and nano chitosan gel (NCG) groups reveal normal histological configurations of hepatic cords (stars) and pancreatic acini (arrowheads). C The hepato-pancreas of the C. albicans group reveals markedly distributed fatty changes (red arrow) and necrotic pancreatic acini with pyknotic nuclei (black arrow). D Hepato-pancreas of the NCG + C. albicans group reveals less number of fatty degenerated cells (arrow) and amelioration in pancreatic acinar epithelium (arrowhead). Scale bar 20 μm

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Additionally, the kidney tissues in the control and NCG groups demonstrated typical histological configurations of renal tubular epithelium, glomerular tufts, and vascular tissue (Fig. 4A & B). In contrast, necrosis of some renal epithelium with the presence of remnant cytoplasmic and nuclear material was obvious in the C. albicans group. In addition, congested renal blood vessels and hypo-cellular glomeruli were noted (Fig. 4C). Conversely, maintenance of renal tissue and few degenerative changes in some renal tubules were in the NCG + C. albicans group (Fig. 4D).

Fig. 4
figure 4

Photomicrographs of kidney sections (H&E). A & B Kidneys of the control and nano chitosan gel (NCG) groups reveal normal histological configurations of renal tubular epithelium (arrows) and glomerular tufts (arrowheads). C Kidney of the C. albicans group reveals necrosis of some renal epithelium with the presence of remnant cytoplasmic and nuclear material (black arrowhead), congested renal blood vessels (red arrow), and hypo-cellular glomeruli (red arrowhead). D Kidney of the NCG + C. albicans group reveals maintenance of renal tissue with few degenerative changes in some renal tubules (arrow). Scale bar 20 μm

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Neurons, glial cells, and neuropil in histology of brain tissues from the control and NCG groups appeared normal (Fig. 5A & B). On the other side, marked areas of vacuolated neuropils and a large number of pyknotic neurons with perineuronal spaces were in the C. albicans group (Fig. 5C). An improvement in most neuronal structures was observed, accompanied by the presence of few areas of neuropil vacuolations and few numbers of pyknotic neurons in the NCG + C. albicans group (Fig. 5D).

Fig. 5
figure 5

Photomicrographs of brain sections (H&E). A & B Brains of the control and nano chitosan gel (NCG) groups reveal normal histological architectures of neurons (arrows), glia cells, and neuropil (stars). C Brain of the C. albicans group reveals a marked area of vacuolated neuropil (arrowhead) and a large number of pyknotic neurons with perineuronal space (arrow). D Brain of the NCG + C. albicans group reveals an improvement in the most neuronal cell body (arrow), neuropil (star) with the presence of few areas of vacuolated neuropil (arrowhead). Scale bar 20 μm

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In addition, the degree of histological damage in the liver, kidney, and brain tissues is shown in Table 4.

Table 4 Scoring system for evaluation of commonly observed lesions in the liver, kidney, and brain tissues of Nile tilapia experimentally infected with C. albicans and treated with nano chitosan gel (NCG) bath for ten days
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Discussion

In aquaculture practices, fungal diseases constitute a major obstacle in culturing fish [65]. C. albicans induces numerous skin ulcers and mortalities in Nile tilapia and African catfish (Clarias gariepinus) [8]. The usage of NGs has displayed aptitude in sustaining fish health besides its role in chelating toxicity [66]. Hence, the present work aims to investigate the influence of waterborne NCG in combating C. albicans in Nile tilapia.

The behavioral changes of an aquatic organism are a result of a variety of physiological and biochemical processes within the body [67]. Herein, we reveal the occurrence of behavioral alterations induced by C. albicans which may be linked to the decline in the brain indicator (AChE) as confirmed in our data. The lower AchE leads to acetylcholine accumulation at synaptic connections. This alters fish locomotor activity and behavior by throwing off the synchronization between neural and muscle connections [68]. Also, it is opined that C. albicans induced oxidative damage in brain tissue resulting in neurological alteration and consequently, behavioral changes. This result was confirmed in our findings by the high level of 8-OHdG which acts as an oxidative DNA damage biomarker. Snarr et al. [69] illustrated the ability of C. albicans to invade the central nervous system by crossing the blood-brain barrier causing neuronal damage. Additionally, the disruption in behavior could be attributed to the negative impact of C. albicans on immune functions. Fish behavioral patterns are linked to the expression of particular cytokines in the brain. Fish with an immune suppression cytokine profile would decrease interaction in several areas of their social behavior and activity and become more vulnerable to predators [70]. Likely, Tartor et al. [5] supported our findings and described that C. albicans induced major clinical picture and post-mortem changes which in turn influence fish immune function and behavior. A study by Gibney and Drexhage [71] supported this assumption which reported that the majority of behavioral disorders are linked to immunosuppression, via altering cytokines levels, and in turn badly influence neuronal function.

Surprisingly, NCG has promise in modulating the neuro indicators and in turn, regenerating the behavioral alterations. It is assumed that the minute size of NCG can reach the brain and exert antioxidant activity which protects the brain tissue from oxidative damage produced by C. albicans. This was validated by the improvement of the histopathological picture of the brain tissues in our results. Concurrent with a recent report [34] describes that NGs have a potential role as nano-carriers for targeting the brain, via crossing the blood-brain barrier and efficiently delivering beneficial agents to the central nervous system. Nano chitosan exerted a neuroprotective potential by boosting the activity of glutathione peroxidase and superoxide dismutase enzymes which can scavenge the produced reactive oxygen species (ROS) hence, reducing oxidative stress [72]. Moreover, another study [73] explained that nano chitosan can protect against neurological damages via conjugation with nerve growth factor, plasmid DNA, and acteoside as well as reversing dopaminergic neuron loss led to amelioration of behavioral disorders.

Assessment of hepato-renal function biomarkers is essential to improve our knowledge about liver and kidney dysfunction in fish especially during infection [74]. Infection by C. albicans resulted in elevating hepatic (ALT, AST, and ALP) and renal indicators (urea and creatinine). This effect could be attributed to the effect of the virulence genes which alter the hepato-renal function via induction of oxidative damage [75]. Similarly, Oda et al. [9] reported an increase in hepato-renal indicators post-exposure of Nile tilapia to C. albicans. It is noteworthy that NCG has a modulating role in the hepato-renal parameters after the C. albicans challenge. Such modulation could be dominated by the antimicrobial role of NCG in eliminating C. albicans infection, and accordingly regenerating hepato-renal function. These findings were also confirmed via noticeable modulation and regeneration of the hepato-renal histological architecture in our findings. In line with previous studies, Ganan et al. [76] and Al-Zahrani et al. [77] described the antimicrobial activity of chitosan nanocomposites and suggested that the existence of amine groups (NH3+) in glucosamine which delivers chitosan its poly-cationic landscape. This nature gives chitosan the power to stick to the negatively charged surface of microorganisms, inducing deviations in the cell surface and consequent outflow of intracellular substances, then cell death.

Exposure of fish to fungal diseases alters the fish’s biochemical traits, including stress indicators [78]. Cortisol, GLU, and NOR are vital stress biomarkers in fish [79]. We reported that GLU, cortisol, and NOR values were elevated in the exposed fish to C. albicans infection reflecting a potent stress response. A recent study by Lemos et al. [80] elucidated the mechanism of induction of stress conditions in fish. First, stress response in fish commences at the cellular level. Such a response may alter the individual homeostasis induce metabolic and physiological changes and possibly cause organismal functional weakening. Primary responses include endocrinological changes, such as increased concentrations of catecholamines (including NOR) and corticosteroids. Secondary responses include metabolic disturbances and elevated GLU levels. Finally, tertiary response involves changes in the behavioral pattern, which is verified in our findings. On the other hand, exposure to NCG modulated the stress indicator levels. It is possible that the minute-sized nano gel effectively diminished the fungal infection via its antimicrobial activity and gradually relieved the stress condition.

In the aquaculture industry, fish infections cause oxidative damage by producing excessive ROS and ultimately contribute to the pathogenesis of pathogens [81]. In the current work, the oxidative damage elicited by C. albicans recorded a notable decline in the activities of TAC and GSH and an increase in the oxidative indicators (MDA and 8-OHdG). This could be dominated by the ability of C. albicans to stimulate the release of more ROS, combined with the stress prompted by the pathogen itself, resulting in suppressing antioxidant activity and augmenting the production of ROS [82]. This finding was reinforced by a recent study that exhibited that C. albicans produced oxidative stress and declined values of catalase and superoxide dismutase in African catfish (Clarias gariepinus) [83]. Astonishingly, NCG applies a strong antioxidant capacity indicated by modifying TAC and GSH levels and reducing the values of MDA and 8-OHdG. This could be attributed to the power of chitosan to successfully hunt ROS and accordingly modulate the activity of antioxidant enzymes. Concurrently, Dizaj et al. [84] designated the antimicrobial activity of nanocomposites which involves the formation of ROS which prompts an elevation in oxidative damage in microbial cells and major damage to biomolecules, inducing genotoxic impacts. In the same instance, Abdel-Tawwab et al. [85] mentioned that the dietary administration of chitosan NPs fortified antioxidant activity in Nile tilapia.

C. albicans adversely altered the hepatic, renal, and brain histological pictures which could be attributed to its virulence capacity. Likewise, a study by Henriques and Silva [86] supported our findings and described the virulence of C. albicans factors which are accountable for the infection. These factors include adherence to the host, the formation of biofilm, and the release of hydrolytic enzymes. Furthermore, C. albicans can colonize on the epithelial surface and grow inducing deep piercing into tissues and severe injury in Zebrafish (Danio rerio) [87]. Similar histopathological changes were reported in the liver and kidney of Nile tilapia by C. albicans [7].

Focusing on the importance of NCG, the study outcomes implied that the NCG-exposed group has a defined improvement in the histological architecture following the challenge. The protecting activity of NCG is opined to its ability to easily penetrate tissues inducing antioxidant and antimicrobial activity which in turn protects tissues from the oxidative damage induced by C. albicans. Also, it could be attributed that NGs are characterized by effective encapsulation and binding activity with the microorganism as clarified by Jiang et al. [88]. Consistent with a recent report, Kuperkar et al. [89] revealed that the small-sized nano gel enables it to overpass the minute capillaries and then pierce tissues through a transcellular pathway. Chitosan can suppress microbial growth via binding with vital minerals and nutrients causing leakage of smaller particles such as phosphate and potassium, followed by larger particles such as DNA and RNA [90]. Furthermore, Chandrasekaran et al. [91] revealed that the antimicrobial action of chitosan nanocomposites is attributed to its metallic ion chelating property.

Based on the study outcomes, NCG has favorable effects on fish performance and can alleviate the negative impacts of C. albicans in Nile tilapia. These outcomes are because of its capability to relieve stress conditions and behavioral changes, modulate hepato-renal and neurological functions, and exert potent antioxidant activity in hepatic tissue, besides maintaining the histological architectures.

Conclusion

The findings of the current study imply the promising effects of the NCG (75 µg/L) in mitigating the hazardous effects of C. albicans infection in Nile tilapia. In addition, NCG is found to possess various privileges including potential neuro-behavioral and stress modulators, antioxidant activity, regenerating hepato-renal function, and histopathological changes induced by C. albicans. Therefore, using NCG as an antimicrobial substitute may favorably sustain the aquaculture sector. Further studies are required to test its efficacy on a broad spectrum of pathogens (both fungi and bacteria) as well as on other farmed aquatic species. In addition to investigating the long-term effects of NCG treatment on fish health and productivity.

Data availability

All data generated or analyzed during this study are included in this article.

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Acknowledgements

This work was supported by the Researches Supporting Project (RSP2025R36), King Saud University, Riyadh, Saudi Arabia. The authors thank the Aquatic Animal Medicine Department, Faculty of Veterinary Medicine, Zagazig University, for their kind help during the experimental procedures.

Funding

This work was supported by the Researches Supporting Project (RSP2025R36), King Saud University, Riyadh, Saudi Arabia.

Author information

Author notes

  1. Heba H. Mahboub and Afaf N. Abdel Rahman contributed equally to this work.

Authors and Affiliations

  1. Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, PO Box 44511, Zagazig, Sharkia, Egypt

    Heba H. Mahboub & Afaf N. Abdel Rahman

  2. Department of Pharmacology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt

    Sara T. Elazab

  3. Department of Zoology, College of Science, King Saud University, PO Box 2455, Riyadh, 11451, Saudi Arabia

    Abdelwahab A. Abdelwarith & Elsayed M. Younis

  4. Department of Pathology, Faculty of Veterinary Medicine, Cairo University, PO Box 12211, Giza, Egypt

    Mohamed Shaalan

  5. Polymer Institute, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, 84541, Slovakia

    Mohamed Shaalan

  6. Department of Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, University of Sadat City, PO Box 32958, Sadat City, Egypt

    Enas K. Aziz

  7. Pathology Department, Faculty of Veterinary Medicine, Zagazig University, PO Box 44511, Zagazig, Sharkia, Egypt

    Mohammed S. Sobh

  8. Department of Veterinary Medicine, RUDN University, Miklukho-Maklaya St, Moscow, 117198, Russia

    Morteza Yousefi

  9. Faculty of Nanotechnology for Postgraduate Studies, Cairo University, Sheikh Zayed Branch Campus, PO Box 12588, Sheikh Zayed City, Giza, Egypt

    Sameh H. Ismail

  10. Aquaculture Nutrition Research Unit ANRU, Carna Research Station, Ryan Institute, College of Science and Engineering, University of Galway, Galway, H91V8Y1, Ireland

    Simon J. Davies

  11. Department of Pathology, Faculty of Veterinary Medicine, Benha University, PO Box 13736, Benha, Toukh, Egypt

    Mohamed M. S. Gaballa

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  1. Heba H. Mahboub
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Contributions

HHM, ANA-conceptualization, methodology, resources, data collection and analysis, visualization, validation, writing original draft, writing– review and editing; STA- conceptualization, investigation, data analysis, writing– review and editing, AAA, EMY-conceptualization, funding acquisition, writing– review and editing, MS, MSS, MMSG-methodology, data collection and analysis, visualization, validation, writing original draft, writing– review, and editing. MY, SJD: writing– review and editing. EKA-methodology, data collection, and analysis, visualization, validation, writing original draft; SHI-methodology, data collection and analysis, validation, writing the original draft. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Heba H. Mahboub or Mohamed Shaalan.

Ethics declarations

Ethics approval and consent to participate

The Institutional Animal Care and Use Committee of Zagazig University, Egypt approved the experimental protocol (ZUIACUC–2-F–333–2022). All methods were performed in accordance with the relevant guidelines and regulations. Informed consent has been obtained from the private farm owners.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Mahboub, H.H., Rahman, A.N.A., Elazab, S.T. et al. Nano-chitosan hydrogel alleviates Candida albicans-induced health alterations in Nile tilapia (Oreochromis niloticus): antioxidant response, neuro-behaviors, hepato-renal functions, and histopathological investigation. BMC Vet Res 21, 159 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04568-4

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04568-4

Keywords

BMC Veterinary Research

ISSN: 1746-6148

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