Chitosan neem nanocapsule enhances immunity and disease resistance in nile tilapia (Oreochromis niloticus)

Abstract

Finding eco-friendly alternatives for antibiotics in treating bacterial diseases affecting the aquaculture sector is essential. Herbal plants are promising alternatives, especially when combined with nanomaterials. Neem (Azadirachta indica) leaves extract was synthesized using a chitosan nanocapsule. Chitosan neem nanocapsule (CNNC) was tested in-vitro and in-vivo against the Aeromonas sobria (A. sobria) challenge in Nile tilapia. A preliminary experiment with 120 Nile tilapia was conducted to determine the therapeutic dose of CNNC, which was established to be 1 mg/L. A treatment study was applied for seven days using 200 fish categorized into four groups (10 fish/replicate: 50 fish/group). The first (control) and second (CNNC) groups were treated with 0 and 1 mg/L CNNC in water without being challenged. The third (A. sobria) and fourth (CNNC + A. sobria) groups were treated with 0 and 1 mg/L CNNC, respectively, and challenged with A. sobria (1 × 10⁷ CFU/mL). Interestingly, CNNC had an in-vitro antibacterial activity against A. sobria; the minimum inhibitory concentration and minimum bactericidal concentration of CNNC against A. sobria were 6.25 and 12.5 mg/mL, respectively. A. sobria challenge caused behavioral alterations, skin hemorrhage, fin rot, and reduced survivability (60%). The infected fish suffered a noticeable elevation in the malondialdehyde level and hepato-renal function markers (aspartate aminotransferase, alanine aminotransferase, and creatinine). Moreover, a clear depletion in the level of the antioxidant and immune indicators (catalase, reduced glutathione, lysozymes, nitric oxide, and complement 3) was obvious in the A. sobria group. Treatment of the A. sobria-challenged fish with 1 mg/L CNNC recovered these parameters and enhanced fish survivability. Overall, CNNC can be used as a new versatile tool at 1 mg/L as a water treatment for combating the A. sobria challenge for sustainable aquaculture production.

1. Introduction

The aquaculture sector has significant importance for millions of people globally, particularly in poor nations where the industry plays a crucial role in job opportunities, economic empowerment, and enhancing the nutritional status of poor people [1]. The consumption of fish as a main source of protein has significantly grown in recent decades among the poorest people [2]. So, many nations are working hard to increase the amount of fish production to meet the consumer's demands [3,4].

Diseased problems cause big mortalities and financial losses, consequently hindering the sustainable development of aquaculture (SDA) [5]. Worldwide, bacterial fish illnesses result in significant economic losses [6]. One of the most prevalent diseases, the bacterial species from the genus Aeromonas, is mostly to blame. Aeromonas spp. Infection severely impacts this fish's global productivity and survival rates [7]. A. sobria is a Gram-negative bacterium that induces great losses in fish farms. Infected fish with A. sobria die from hemorrhagic septicemia, ascites, and ulcer development. Extracellular hemolysin and aerolysin synthesis are two important virulence genes responsible for developing septicemia [8].

Treating bacterial diseases in aquaculture with antibiotics leads to the antibiotic resistance hazard, which risks consumer well-being [9]. Using antibiotics to treat aquatic animals can result in several difficult issues, including the emergence of antibiotic-resistant bacterial strains, antibiotic remnants present in the fish muscles that impact the consumers' well-being, and environmental pollution [10,11]. To minimize the harmful effects of antibiotic usage, developing promising, safe, ecologically friendly alternatives is vital [12].

The herbal plants are a safe and eco-friendly alternative to antibiotics. The majority of molecules derived from plants are phytochemicals and secondary metabolites, which are categorized into a variety of classes and mostly function as antimicrobials such as tannins, coumarins, terpenes, quinones, phenolics, polyphenols, flavonoids, lectins, polypeptides, and saponins [13,14].

Neem (Azadirachta indica) is a large evergreen tree with fragrant foliage and tasty fruits. This tree's anti-inflammation, immunological, and anti-ulcer properties have made it widely employed for various purposes [15,16]. Additionally, every part of the neem tree possesses various antifungal, antibacterial, and antiviral properties and antioxidant properties [17,18]. The neem leaves had antibacterial activity against the Staphylococcus aureus and A. hydrophila isolated from singhi (Heteropneustes fossilis) and mrigal (Cirrhinus mrigala) [19]. Neem leaves extract had antibacterial activity against A. veronii, A. hydrophila, Acinetobacter junii, A. tandoii, and Pseudomonas stutzeri isolated from blackspot barb (Dawkinsia filamentosa) [20].

As a superior alternative to conventional methods that rely on the preparation of pharmaceutical drugs in biomedical applications, novel advances in nanotechnology have aroused the interest of researchers in a variety of scientific fields and aquacultures. Examples of novel advances are nanostructured materials such as nanoparticles, conjugates, and nanocapsules [21]. Nano-medicine uses various nanotechnology tools to create quicker and more effective treatments for illnesses or disease management [22]. One of the materials employed the most frequently in nanotechnology is chitosan, a degradable polysaccharide with various applications [23,24]. Because nanostructured materials may amplify the action of plant extracts, decreasing the needed dose and adverse effects, and enhancing activity, employing the nanocapsule formulation has numerous benefits over using the herbal addition. This is because it has been widely advocated to combine herbal medicine with nanotechnology. Throughout the treatment period, the active component can be administered by nanosystems at an appropriate concentration, sending it to the intended action location. Traditional medical procedures fall short of these requirements [25].

Recently, neem leaves extract was loaded on chitosan nanoparticles forming nanocomposites which exhibited an inhibitory effect against pathogenic Gram-negative and Gram-positive bacterial strains in the bacterial biofilm [26]. We designated this study based on the severe impacts of A. sobria on Nile tilapia (Oreochromis niloticus) health and productivity and the hazards of antibiotics usage in aquaculture on fish health and, consequently, consumers' well-being. The current investigation was intended to investigate the probable therapeutic effect of chitosan neem nanocapsule (CNNC) as an alternative to antibiotics against A. sobria-induced hepato-renal dysfunction, antioxidant, and immune suppression in Nile tilapia.

2. Materials and methods

2.1. CNNC preparation

The fresh neem leaves were provided from the Desert Research Center, Gizza, Egypt. The leaves were air-dried and mixed using a Moulinex mixer (Type 716, France) to obtain the powder form. For the preparation of the extract, about 10 g of the powdered neem leaves were soaked in 250 mL of doubly deionized water and sonicated for 1 h at 50 kHz and cycle 0.6 using a Hielscher UP400S sonicator prop apparatus. The extract was processed using Whatman No. 1 filter paper. Then, CNNC was prepared by the sono-chemical method [27] with some modifications. Briefly, chitosan (Sigma-Aldrich) aqueous solution (0.2% w/v) was mixed with an acetic acid solution (2% v/v) at 60 °C and stirred for 1 h. Then, about 50 mL of neem extract was added with vigorous stirring, and finally, sodium tripolyphosphate (TPP) solution (1% w/v) was added drop by drop until a green-whitish mixture appeared. The mixture was washed in a beaker of 2 L deionized water and let to be precipitated, and then the supernatant solution was removed. The final steps were repeated three times to remove any access to secondary products from the synthesis method.

2.2. Characterization of the CNNC

The X-ray diffraction (XRD, Thermo Scientific company model of EQUINOX 1000) was used to determine the CNNC composition to be sure that the obtained CNNC was formed without any secondary chemicals from synthesis. Characterization of the CNNC morphology was performed by transmission electron microscopy (TEM, JEOL company model of JEM-2100 high-resolution) and atomic force microscopy (AFM, Agilent technology company model of 5600LS). Specific surface area [Brunauer–Emmett–Teller (BET) method] and pore size [Dollimore and Heal (DH) method ] were determined using a surface area and pore size analyzer (Quanta Chrome Company model of Nova Touch 4 L). Size (Dynamic light scattering, DLS) and charge (zeta potential) were determined using a zeta seizer (Malvern Company model of Nano Sight NS500).

2.3. Bacterial strain

The A. sobria strain from dead fish was previously found by the Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, and it was verified that it was harmful to O. niloticus [[28], [29], [30], [31]]. Both standard biochemical assays and the VITEK® 2 system (BioMérieux Inc., NC, USA) were used to identify the isolate. After the bacterial isolate was cultivated for 24 h at 27 °C on tryptic soy agar, one colony was selected to incubate for 24 h at that temperature in tryptic soy broth. The pellet from the cultured broth containing the isolates was extracted by centrifuging at 3000 rpm for 10 min at 4 °C. The pellet was then suspended in a sterile phosphate-buffered saline solution.

2.4. In-vitro antibacterial activity

The antibacterial activity of CNNC and chitosan against A. sobria was tested using the disc diffusion assay [32]. The bacterial isolate's overnight culture was applied on Muller Hinton agar (Difco Laboratories). Once the turbidity matched McFarland tube number 0.5 (1.5 × 10⁸ CFU/mL), pure colonies of the bacterium were suspended in sterile saline and swabbed onto Muller Hinton agar in a loopful. Different concentrations of CNNC and chitosan were used (0, 6.25, 12.5, 25, 50, and 100 mg/mL), and the sterile paper discs (6 mm in diameter) were dried at 100 °C for 2 h in a hot air oven before being applied to the inoculated agar plates. Then, the zones of inhibition were measured in mm on the plates after they had been incubated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays for CNNC and chitosan were determined. The MIC was carried out [33] with some changes. Different CNNC and chitosan concentrations (0, 6.25, 12.5, 25, 50, and 100 mg/mL) were added to a 96-well microtitre plate, and each concentration was added in a separate well. Then, about ten μL of each standardized broth culture (1.5 × 10⁸ CFU/mL) were added to each well. The microtitre plate was then incubated following organism growth requirements, and any audible bacterial growth was checked. The MIC was the lowest CNNC and chitosan concentration at which there was no observable growth on the agar surface. MBC was done by sub-culturing from each well of the microplate onto a nutrient agar plate. The well containing the least concentration of the CNNC and chitosan that did not produce growth on subculture was regarded as MBC for that test strain.

2.5. Fish and rearing conditions

Four hundred and twenty cultured O. niloticus (25 ± 2.10 g) that seemed in good condition were acquired for the in-vivo test from the Fish Research Unit at the Faculty of Veterinary Medicine at Zagazig University of Egypt. Fish were observed and acclimated for two weeks before the trial. An assessment of the fish's health was also done to ensure they were well. Ten fish were housed in glass aquariums (40 × 30 × 80 cm) with 60 L of dechlorinated tap water. The water criteria measurements, which included dissolved oxygen (6.00 ± 0.50 mg/L), pH (6.3 ± 0.6), temperature (26 ± 1.2 °C), and ammonia (0.030 ± 0.01 mg/L), were established with a controlled day to night period (12 h dark: 12 h light), following the APHA guidelines [34] to be suitable for Nile tilapia culture. A basal diet of 3% was fed to the fish. The animal usage in the research committee at Zagazig University in Egypt approved the experimental protocol (ZU-IACUC/2/F/310/2022).

2.6. Determination of CNNC therapeutic dosage

To determine the therapeutic dose of CNNC, the method of Abdel Rahman et al. [35] was applied with some modifications. About 120 fish were exposed to 12 grading levels of CNNC (0, 0.5, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 4.50, 5.50, and 6 mg/L) for 7 days (Table 1). Daily monitoring for the mortalities and observable signs was carried out throughout the exposure period (7 days). The safe concentration of CNNC for the therapy study was 1 mg/L.

Table 1.

Effect of the exposure to different concentrations of CNNC on mortality and clinical observations of O. niloticus for seven days.

Conc. (mg/L)		Clinical observations
	Mortality (N = 10)	Loss of escape reflex	Abnormal swimming	External skin lesion
0.0	0/10	–	–	–
0.5	0/10	–	–	–
1	0/10	–	–	–
1.5	1/10	–	–	–
2	1/10	–	–	–
2.5	2/10	–	–	–
3	2/10	–	–	–
3.5	3/10	+	+	+
4	3/10	++	++	++
4.5	3/10	++	++	++
5	4/10	+++	+++	+++
5.5	4/10	+++	+++	+++
6	4/10	+++	+++	+++

(−) no abnormal observations; (+) mild abnormal observations; (++) moderate abnormal observations (+++); severe abnormal observations.

2.7. Experimental setup

The lethal dose 50 (LD₅₀) of A. sobria was calculated [29] as follow; a total of 100 fish were distributed into five groups in two replication (20 fish/group; 10 fish/replicate). Fish were fasted for 24 h before the experimental infection. The intraperitoneal (IP) injection of the first to the fourth groups was performed using 0.5 mL of dosages of A. sobria bacterial suspension (10⁶, 10⁷, 10⁸, and 10⁹ CFU/fish). About 0.5 mL of sterile saline was IP injected into the fifth group. The mortality of the infected fish was tracked for four days following injection. The LD₅₀ was computed as 2 × 10⁷ CFU/mL using the Probit Analysis Program version 1.5.

The challenge test was employed using a sub-lethal dosage of 1 × 10⁷ CFU/mL, and the outcomes were verified using a drop plate test [36]. A total of 200 fish were randomly divided into four groups, with 50 fish per group in five repetitions over seven days. The first (control) and second (CNNC) groups received treatments of 0 and 1 mg/L CNNC in water without being challenged. The third and fourth groups (A. sobria and A. sobria + CNNC, respectively) received 0 and 1 mg/L CNNC treatments and were IP challenged with 0.2 mL of A. sobria (1 × 10⁷ CFU/mL). Throughout the seven-day study, the clinical symptoms and post-mortem findings were noted. The fish survival rate (%) was determined using Eq. (1).

$$\mathrm{S}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}(\%)=100\times \frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{s}\mathrm{h}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{m}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l}\left(7\mathrm{d}\mathrm{a}\mathrm{y}\mathrm{s}\right)}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{s}\mathrm{h}}$$ (1)

2.8. Blood sampling

Fish were anesthetized with 100 mg/L benzocaine solution [37] at the termination of the experiment (7 days). The caudal vessels were used to collect blood samples (15 fish/group), which were then centrifuged at 4 °C for 10 min to separate the serum at 3000 rpm, and then kept at 20 °C pending the immunological and biochemical testing.

2.9. Serum antioxidant/oxidant assays

According to the Ohkawa et al. [38] approach, the malondialdehyde (MDA) level was determined using the thiobarbituric acid assay. The thiobarbituric acid reactive compounds were detected at 535 nm, and the level of MDA was then determined. According to the technique described by Aebi [39], catalase (CAT) activity was measured, and the enzymatic reaction mixture used in this assay included potassium phosphate (pH 7.0), hydrogen peroxide (H₂O₂), and homogenate supernatant. Using a UV-VIS spectrophotometer, the molar attenuation coefficient of H₂O₂ was investigated at 240 nm.

The CAT amount degrading H₂O₂ (1 μmol)/minute/milligram of hippocampal tissue protein (U/mg protein) was used to measure CAT activity. Reduced glutathione content (GSH) was measured calorimetrically following the method of Beutler [40]. A spectrophotometric test at 412 nm of the yellow derivative produced by the reaction of the supernatant with 5-5′ dithiobis-2-nitrobenzoic acid and protein precipitation by meta-phosphoric acid are both used in this method. Glutathione activity was assayed by monitoring glutathione utilization by H₂O₂.

2.10. Hepato-renal-functions-related assays

The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were estimated by Spinreact kits (Esteve De Bas, Girona, Spain) at 540 nm following the Burits and Ashwood [41] and Reitman and Frankel [42] protocols. Creatinine was estimated by Spinreact kits (Esteve De Bas, Girona, Spain) following the Fossati et al. [43] approach.

2.11. Immune-related assays

A colorimetric test was followed to evaluate the nitric oxide (NO) level following Montgomery and Dymock [44] technique by detecting nitrite (NO₂-) using Griess reagent and converting nitrate (NO₃-) to NO₂- using vanadium chloride as a reducing agent. To remove undesirable proteins from the sample, 1 mL of 100% alcohol and 0.5 mL of serum sample were combined, and the mixture was then centrifuged at 3000 rpm for 20 min. Next, 0.5 mL of the produced sample was added with 0.5 mL of the Griess reagent and 0.5 mL of vanadium chloride (0.8%) in 1 M HCl. The absorbance was measured at 540 nm against deionized water following thorough mixing and incubation for 30 min At 37 °C. To create a standard curve, various sodium NO₂-concentrations were employed.

The serum lysozyme (LYZ) activity was assessed using spectrophotometry [45] based on the lysis of Micrococcus lysodeikticus (Sigma Co., USA) with some changes. The serum and M. lysodeikticus suspension (0.2 mg/mL in 0.05 M PBS, pH 6.2) were combined at 25 °C for 5 min. The optical density was evaluated at 540 nm for 5 min at 1-min intervals. To determine the serum LYZ level, a calibration curve was done using various concentrations of lyophilized chicken egg-white lysozyme (Sigma Co., USA). Serum complement 3 (C3) was measured using MyBioSource Co. ELISA kits (Cat. No. MBS005953), and this included measuring the rise in turbidity after the immune system's reaction to the anti-C3 antibody at 340 nm.

2.12. Data analysis

To assess the treatment impact of CNNC against A. sobria, the data were first submitted to a normality test (the Kolmogorov-Smirnov) and then subjected to a two-way ANOVA using the SPSS program (version 20; Richmond, VA, USA). Duncan's post hoc test was performed to evaluate mean differences using the 5% probability level. The findings were shown as means ± standard error. The LD₅₀ of A. sobria was determined using the Probit Analysis Program, version 1.5, of the US Environmental Protection Agency.

3. Results

3.1. Characterization of the CNNC

As illustrated in Fig. 1A, the XRD pattern confirmed the amorphous nature of CNNC. TEM image showed the sub-spherical shape of CNNC without any aggregation or agglomeration (Fig. 1B). AFM images (3D and 2D) illustrated the triangle shape to the sub-spherical nature of CNNC with different particle size distributions (Fig. 1C and D).

Fig. 1.

The characterization patterns of CNNC: (A) XRD, (B) TEM, and (C and D) AFM 3 D and 2 D.

The surface area estimated with the BET approach clearly showed how highly valued CNNC was. The pore volume established by the DH technique was 1.2 cc/nm, and the BET surface area value was 74.2 m²/g. Fig. 2A illustrates the isotherm of CNNC, which have type II isotherm. Fig. 2B reveals the microporous using the DH method. DLS exhibited that the particle size and distribution were 24 and 55 nm (Fig. 2C). Zeta potential value was −30 mV (Fig. 2D).

Fig. 2.

The characterization patterns of CNNC: (A) Isotherm, (B) pore size and pore volume according to the DH method, (C) DLS, and (D) Zeta potential.

3.2. In-vitro antibacterial activity

CNNC and chitosan showed antibacterial activity against A. sobria with different inhibition zone diameters depending on the concentrations. At the concentrations of 0, 6.25, 12.5, 25, 50, and 100 mg/mL, the inhibition zones of CNNC were 0, 2.5, 6.3, 9.23, 12, and 16 mm (Fig. 3 A) for CNNC and were 0, 0, 0, 1.85, 2.20, and 3 mm (Fig. 3B) for chitosan, respectively. The MIC of CNNC and chitosan against A. sobria were 6.25 and 100 mg/mL, respectively. Meanwhile, the MBC of CNNC and chitosan were 12.5 and 100 mg/mL, respectively (Table 2).

Fig. 3.

Inhibition zones (mm) against A. sobria at a concentration of 100 mg/mL of CNNC (A) and chitosan (B).

Table 2.

The inhibition zone diameter (mm), MIC, and MBC of CNNC and chitosan against A. sobria.

3.3. Clinical signs, behaviors, and survivability

The fish of the control and CNNC groups exhibited the highest survivability (100%). They showed no abnormal behaviors (Table 3) or symptoms (Fig. 4A). The survival rate of A. sobria group fish was low (60%). They also had fin rot, hemorrhages on various body parts, and skin ulcerations (Fig. 4B and C) with internal organs congestion. Likewise, the CNNC + A. sobria group showed increased survivability (85%) and alleviation of the previous disease signs, with only slight fin rot of the tail fin (Fig. 4D).

Table 3.

Effect of A. sobria infection and/or CNNC as water treatment on behavioral responses and survival rate (%) of O. niloticus for seven days.

Parameters	Control	CNNC	A. sobria	CNNC + A. sobria
Surfacing	–	–	+++	+
Abnormal swimming	–	–	+++	+
Loss of escape reflex	–	–	+++	+
Survival rate (%)	100	100	60	85

The score of symptoms was recorded as (−) no, (+) mild, (++) moderate, and (+++) severe.

Fig. 4.

Effect of CNNC as water exposure on clinical observations of experimentally infected O. niloticus with A. sobria for seven days. (A) Fish of the control group or CNNC group demonstrating normal appearance. (B and C) Fish of the A. sobria group demonstrating skin ulcerations (blue arrows), body hemorrhages, and fin rot (yellow arrows). (D) Fish of the CNNC + A. sobria group demonstrating slight fin rot (yellow arrow).

3.4. Antioxidant/oxidant parameters

In comparison with the non-infected groups, the antioxidant enzymes (CAT and GSH) were considerably lower (P < 0.05), and the MDA level was considerably higher (P < 0.05) in the infected groups. Regarding the impact of CNNC water treatment, CNNC treatment (1 mg/L) increased the GSH level and decreased the MDA level (P < 0.05) but had no impact (P > 0.05) on the CAT level.

Concerning the interaction between the A. sobria infection and CNNC water application, the non-infected group and treated with 1 mg/L CNNC showed a significantly (P < 0.05) lower level of MDA and higher level of CAT and GSH (P < 0.05) than the non-infected one and treated with 0 mg/L CNNC. However, the infected group treated with 0 mg/L CNNC had a substantial decline (P < 0.05) in the levels of CAT and GSH and a significant increase (P < 0.05) in the level of MDA. When the infected group received 1 mg/L CNNC treatment, these parameters were dramatically altered and improved (Table 4).

Table 4.

Effect of A. sobria infection and/or CNNC as water treatment on the oxidant/antioxidant biomarkers of O. niloticus for seven days.

A. sobria infection	CNNC (mg/L)	MDA (nmol/mL)	CAT (ng/mL)	GSH (ng/mL)
Effect of A. sobria infection
Non-Infected		1.42 ± 1.96b	154.22 ± 9.94a	162.46 ± 5.63a
Infected		12.85 ± 3.12a	30.80 ± 2.85b	33.10 ± 1.66b
Effect of CNNC as water treatment
	0	11.01 ± 1.96a	86.29 ± 7.58	72.46 ± 5.25b
	1	3.26 ± 1.32b	98.73 ± 9.94	123.09 ± 6.85a
Interaction
Non-infected	0	1.14 ± 0.04c	152.15 ± 3.65a	133.90 ± 3.04b
	1	1.71 ± 0.01c	156.30 ± 2.35a	191.02 ± 2.96a
Infected	0	20.89 ± 0.32a	20.43 ± 0.44c	11.03 ± 0.49d
	1	4.82 ± 0.17b	41.17 ± 1.85b	55.16 ± 0.93c
Two-way ANOVA	P-value
A. sobria infection		0.001	0.01	0.02
CNNC water treatment		0.01	0.39	0.01
Interaction		0.02	0.001	0.001

MDA, malondialdehyde; CAT, catalase; GSH, reduced glutathione. The values were expressed as mean ± SE (N = 15/group). Values in the same column that did not share the same superscripts differed significantly (P < 0.05; two-way ANOVA).

3.5. Hepato-renal functions-related parameters

In comparison with the non-infected groups, the levels of the hepato-renal function markers (AST, ALT, and creatinine) were substantially higher in A. sobria-infected groups (P < 0.05) than the control one. Regarding the CNNC water treatment impact, the liver function indicators (AST and ALT) were considerably decreased (P < 0.05) by treatment with 1 mg/L CNNC in comparison to the non-treated groups, with no significant influence (P > 0.05) on creatinine level.

Concerning the interaction between A. sobria infection and CNNC water application, it was noticed that the hepato-renal function indicators were elevated significantly (P < 0.05) in the infected group, which was treated with 0 mg/L CNNC followed by the infected one, which was treated with 1 mg/L CNNC compared to other groups (Table 5).

Table 5.

Effect of A. sobria infection and/or CNNC as water treatment on the hepato-renal biomarkers of O. niloticus for seven days.

A. sobria infection	CNNC (mg/L)	AST (U/L)	ALT (U/L)	Creatinine (mg/dL)
Effect of A. sobria infection
Non-infected		84.87 ± 2.01b	16.65 ± 3.15b	0.25 ± 0.04b
Infected		113.72 ± 4.25a	36.68 ± 6.85a	0.36 ± 0.09a
Effect of CNNC as water treatment
	0	104.07 ± 2.01a	32.40 ± 3.15a	0.31 ± 0.04
	1	94.52 ± 2.11b	20.93 ± 2.11b	0.30 ± 0.02
Interaction
Non-infected	0	85.74 ± 0.98c	15.81 ± 1.12c	0.23 ± 0.01c
	1	84.00 ± 2.01c	17.50 ± 1.50c	0.27 ± 0.02c
Infected	0	122.40 ± 2.50a	49.00 ± 2.12a	0.40 ± 0.20a
	1	105.05 ± 3.21b	24.37 ± 1.81b	0.33 ± 0.11b
Two-way ANOVA	P-value
A. sobria infection		0.01	0.001	0.01
CNNC water treatment		0.04	0.001	0.82
Interaction		0.001	0.03	0.01

AST, aspartate aminotransferase; ALT, alanine aminotransferase. The values were expressed as mean ± SE (N = 15/group). Values in the same column that did not share the same superscripts differed significantly (P < 0.05; Two-way ANOVA).

3.6. Immune functions parameters

Comparable to the non-infected groups, the immunological parameters (NO, LYZ, and C3) were substantially lowered (P < 0.05) in the infected groups. These immunological indices were considerably enhanced (P < 0.05) by treatment with 1 mg/L CNNC. In terms of the relationship between the A. sobria infection and CNNC water treatment, it was noticed that the immunological parameters were considerably improved (P < 0.05) in the non-infected groups treated with 1 mg/L CNNC, followed by the non-infected group treated with 0 mg/L CNNC. In contrast, these immunological markers were significantly reduced (P < 0.05) in the infected group treated with 0 mg/L CNNC. When the infected group received 1 mg/L CNNC treatment, these parameters dramatically improved (Table 6).

Table 6.

Effect of A. sobria infection and/or CNNC as water treatment on the immunological biomarkers of O. niloticus for seven days.

A. sobria infection	CNNC (mg/L)	NO (ng/mL)	LYZ (ng/mL)	C3 (mg/dL)
Effect of A. sobria infection
Non-infected		406.11 ± 15.12a	5.88 ± 1.12a	11.67 ± 2.25a
Infected		177.92 ± 6.43b	1.04 ± 0.11b	3.34 ± 0.45b
Effect of CNNC as water treatment
	0	269.41 ± 4.22b	2.41 ± 0.11b	5.51 ± 0.45b
	1	314.62 ± 6.43a	4.51 ± 0.24a	9.49 ± 2.15a
Interaction
Non-infected	0	371.23 ± 10.13b	4.60 ± 0.20b	9.23 ± 0.49b
	1	441.00 ± 9.10a	7.16 ± 0.10a	14.11 ± 2.00a
Infected	0	167.60 ± 2.47d	0.23 ± 0.02d	1.80 ± 0.10d
	1	188.25 ± 2.01c	1.86 ± 0.10c	4.88 ± 0.02c
Two-way ANOVA	P-value
A. sobria infection		0.01	0.02	0.001
CNNC water treatment		0.01	0.01	0.02
Interaction		0.03	0.001	0.01

NO, nitric oxide; LYZ, lysozyme; C3, complement 3. The values were expressed as mean ± SE (N = 15/group). Values in the same column that did not share the same superscripts differed significantly (P < 0.05; Two-way ANOVA).

4. Discussion

New alternatives to antibiotic therapy should be established to address the problem of antibiotic resistance in aquaculture. This study proposed a novel antibacterial agent for future application in treating bacterial fish pathogens (A. sobria) using the nanotechnology procedure. Synthesis and characterization of CNNC were studied. Nano-encapsulation is a newly developed method used extensively in the pharmaceutical, chemical, cosmetics, and food processing industries [46]. A similar preparation method to the aqueous extract of Ocimum basilicum using the chitosan nanocapsule has been investigated [28]. The CNNC was characterized using several techniques [47], which confirmed the synthesis process of the nano form. The XRD pattern in this study confirmed the amorphous nature of CNNC with a good coating of neem extract by the chitosan nanocapsule. AFM and TEM techniques confirmed the topography and particle shape of CNNC, which revealed the sub-spherical shape without any aggregation or agglomeration of CNNC. In addition, the BET surface area (74.2 m²/g) and pore volume (1.2 cc/nm) for the CNNC indicate improved bioactivity as a result of improving chemical activity. Triangle-shaped particles with two distinct dimensions may be responsible for CNNC's 24 and 55 nm DLS patterns. The high solubility of CNNC in an aqueous solution and negative charge resulting from the effective coating of neem extract, where chitosan has a positive charge, is explained by the high zeta potential value (−30 mV).

Herein, CNNC had an in-vitro antibacterial activity against A. sobria. The antibacterial activity of chitosan-neem nanocomposites against Escherichia coli and S. aureus had been investigated in a previous report [48]. The antibacterial activity of the CNNC against A. sobria could be related to the synergistic effect of both neem leave extract and chitosan (both are antibacterial agents) [[48], [49], [50], [51], [52], [53], [54]]. Moreover, the phytochemical constituents of the neem leaves, such as steroids, glycosides, alkaloids, triterpenoids, saponins, and triterpenes, have antibacterial properties [55]. These phytoconstituents are considered the antibacterial components against pathogens [56]. Also, they have a variety of effects on the microbial cell through different mechanisms, including alterations in the structure and operation of cell membranes [57], interfering with the metabolism of intermediaries [58], disruption of DNA/RNA function and synthesis [59], and quorum sensing, which prevents normal cell communication [60]. Additionally, cytoplasmic components' induction of coagulation [61]. In addition, chitosan interacts with surface molecules and blocks mRNA by attaching to bacterial DNA [62]. According to Raafat et al. [63] and Ganan et al. [64], the amine groups (NH₃ ⁺) of glucosamine in chitosan, which may be a crucial component in its ability to interact with a negatively charged surface element of many microorganisms and cause extensive surface alterations that result in the leakage of intracellular substances and cell death, are what give chitosan its antibacterial potential.

The A. sobria-infected fish suffered hemorrhagic skin, fin rot, decreased swimming behavior, and lowered survivability (60%). Similar outcomes were recorded in previous investigations [[65], [66], [67]]. According to Janda and Abbott [68], these repercussions may be caused by the circulation of bacteria in the blood and some virulence factors, such as hydrolytic enzymes, hemolytic toxins, and cytotoxic enterotoxins. On the other hand, therapy with 1 mg/L CNNC for the A. sobria-infected group restored the prior clinical symptoms and improved fish survivability (85%). These outcomes may be explained by CNNC's antibacterial characteristics, demonstrated in this work by the in-vitro assay.

An imbalance between antioxidant enzyme activity and excessive reactive oxygen species (ROS) production causes oxidative stress [[69], [70], [71]]. Fish are protected from oxidative stress by an enzymatic antioxidant system [72]. Oxidative stress resulted in elevated lipid peroxidation levels, which is indicated by the increase in the level of MDA [70,73,74]. In this regard, the A. sobria-infected group in the current study had lower levels of CAT and GSH but greater levels of MDA. These consequences result from increasing the cell wall lipid peroxidation (MDA) and ROS production by the virulence factors of the bacteria, which are blamed on a state of oxidative stress [75].

Surprisingly, treatment of the A. sobria-infected group with 1 mg/L CNNC modulates these variables. This could be attributed to the prevention of disease progression. Moreover, the synergistic effect of both neem leaves extract and chitosan. The antioxidant properties of neem leaves extract could be attributed to its phytochemical constituents of flavonoids and phenolics, which act as antioxidants to defend against free radicals that can harm tissues and cells [55,76]. Additionally, the chelating activity of chitosan and the free radical scavenging of its hydroxyl function group, which was accomplished by donating two electrons, might be responsible for its antioxidant characteristics [77].

Increased hepato-renal function indicators in the serum indicate liver and kidney dysfunctions [[78], [79], [80]]. A. sobria-infected group showed disruption in the hepato-renal functions (higher AST, ALT, and creatinine) comparable to the control group. A. sobria may produce toxic compounds that could injure hepatocytes and renal cells, increasing these indicators [81]. Modulation of the hepato-renal function indicators of the infected fish was noticed by treatment with 1 mg/L CNNC. These results could be attributed to the antioxidant activity of CNNC, which was evident in this study by decreasing the MDA and increasing the antioxidant enzymes, which help to maintain the tissue health and integrity, accordingly maintaining their functions.

The immune function biomarkers were investigated in the current study. The potent elements of fish humoral non-specific defensive systems include NO [82]. Bacteriolytic enzymes called LYZ are thought to be a sign of a generalized immune response to microbial invasion [83]. The complement system is a group of proteins communicating with the innate and adaptive immune systems in fish and other vertebrates. In the humeral component of innate immunity, the complement system is a noticeable and significant player [84]. Immune function markers (NO, LYZ, and C3) decreased in the A. sobria-infected fish; this outcome may be attributed to the bacteria's pathogenicity. According to earlier studies [81], A. sobria's virulence factors enable the bacteria to damage host tissues and impair host defense mechanisms.

On the contrary, treatment of the A. sobria-infected group with 1 mg/L CNNC enhanced the immunological parameters. These results could be attributed to the combined effect of neem leaves extract and chitosan (both had immune-enhancing properties). A previous study reported that neem leaves extract enhanced the immune system and increased the survivability of Asian seabass (Lates calcarifer) fingerlings challenged with Vibrio harveyi infection [85]. Moreover, dietary chitosan was reported to increase the non-specific immune response of the olive flounder (Paralichthys olivaceus) and improve the water quality [86]. The immuno-stimulant effect of neem extract could be attributed to its constituent, β-sitosterol, which was suggested to have a role in strengthening the immune system [55]. The immune-stimulant properties of chitosan through increasing immunoglobulin M and LYZ levels were previously reported in O. niloticus [87,88].

5. Conclusion

According to the outcomes of the disc diffusion and MIC tests, CNNC exhibits a stronger antibacterial activity against A. sobria. CNNC therapy by 1 mg/L as a water additive can alleviate the health disorders, hepato-renal disruption, and oxidant-immune dysfunction caused by the A. sobria infection. CNNC can be used as a potential alternative to antibiotics in aquaculture for controlling the A. sobria bacterial infection, hence maximizing the aquaculture industry. Future research is required to determine how CNNC affects fish health status at the molecular and histological levels. Also, more research should be designated for testing the antibacterial activity of CNNC against a wide range of fish pathogens.

Author contribution statement

Rowida E. Ibrahim, Gehad E. Elshopakey, Abdelwahab A. Abdelwarith, Elsayed M. Younis, Sameh H. Ismail, Amany I. Ahmed, Mahmoud M. El-Saber, Ahmed E. Abdelhamid, Simon J. Davies, Abdelhakeem El-Murr, and Afaf N. Abdel Rahman: Conceived and designed the experiments, analyzed and interpreted the data, and contributed reagents, materials, analysis tools or data. Rowida E. Ibrahim and Afaf N. Abdel Rahman: Wrote the paper. All authors have made substantial contributions to all of the following: (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for important intellectual content, (3) final approval of the version to be submitted.

Data availability statement

Data will be made available on request.

Additional information

No additional information is available for this paper.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.