Therapeutic effects of white poplar (Populus alba) leave extract on hepatorenal, stress, and antioxidant-immune parameters of Oreochromis niloticus challenged with Aeromonas veronii

Abstract

Herbal plants have gained enormous interest due to their immune, antioxidant, and antibacterial activities. The present study proposed that white poplar (Populus alba) leave extract (WPE) might provide a promising alternative to traditional antibiotics to treat Aeromonas veronii infection in Nile tilapia (Oreochromis niloticus). WPE showed an in vitro antibacterial activity [22 ± 0.45 mm inhibition zone, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 60 µg/mL] against A. veronii. For the in-vivo study, a factorial (2 × 2) design was used to investigate the therapeutic effects of WPE on the antioxidant/immune status and blood biochemical parameters of Nile tilapia challenged with A. veronii. One-hundred sixty fish (33 ± 1.5 g) were assigned into four experimental groups, each with four replicates (4 glass tanks/group, 40 fish/group, 10 fish/tank) for 10 days. In the first (1st) and 2nd groups, 0 and 1.5 mg/L WPE were applied in tank water, respectively, without fish being challenged. In the 3rd and 4th groups, 0 and 1.5 mg/L WPE were applied in tank water, respectively, with fish intraperitoneal inoculated with 0.2 mL of A. veronii (0.5 × 10⁷ CFU). Aeromonas veronii infection significantly diminished the survivability, hepatic catalase, lysozyme activity, nitric oxide, immunoglobulin M, phagocytic %, total protein, albumin, and globulin. Moreover, a significant rise in the hepatic malondialdehyde, serum hepatorenal function indicators, cortisol, and glucose levels were consequences of A. veronii challenge. Interestingly, the interaction between bacterial challenge and WPE application increased the survivability, antioxidant activity, and immune responses and reduced ALT levels of fish treated with WPE during the bacterial challenge. The serum creatinine, cortisol, and glucose levels were decreased in fish treated with WPE during the infection but still higher than in the non-infected fish either treated or not treated with WPE. In conclusion, WPE (1.5 mg/L) can be used as an antibacterial substance in fish farming to alleviate the detrimental impacts of A. veronii infection by enhancing the antioxidant/immune status of the infected fish. These outcomes could help reduce antibiotic usage in fish farms, which is conducive to the sustainable development of aquaculture.

HIGHLIGHTS

• White poplar extract (WPE) had an in vitro antibacterial activity against Aeromonas veronii.

• Aeromonas veronii infection inhibited the antioxidant/immune functions of Nile tilapia.

• Aeromonas veronii infection altered the hepatorenal functions and biochemical parameters of Nile tilapia.

• WPE at a level of 1.5 mg/L displayed a therapeutic effect in Nile tilapia against A. veronii.

Keywords:

• Aeromonas veronii
• antibacterial activity
• antioxidant-immune status
• hepato-renal function
• Populus alba

Introduction

Today, aquaculture produces more than 49.2% of all fish consumed globally (FAO 2022). The speedy growth of fish production results in numerous dangerous problems, such as high-density culture incidents and the shortage of effective disease prevention approaches. Consequently, this extension may initiate stressful conditions, suppressing the immune system and increasing the fish’s vulnerability to infectious agents (Harikrishnan et al. 2011a; Lieke et al. 2020). In addition, various stressors that result from environmental changes and human activity can pose problems for fish (Schreck and Tort 2016; Reid et al. 2019). Fish immune systems and antioxidant capability are compromised by these stressors, which causes reproductive dysfunction and potentially catastrophic financial losses (Ahmed et al. 2022).

Treatment of bacterial pathogens using antibiotics in aquaculture is not endorsed due to their residues in fish, increased resistant pathogenic bacteria, and adverse environmental impacts that harm human well-being (El-Nobi et al. 2021). Among these pathogens was the Aeromonas veronii (A. veronii) infection, which causes ulcerative lesions and haemorrhagic septicaemia (Amal et al. 2018; Abd El Latif et al. 2019; Youssuf et al. 2020). The significant threats behind A. veronii infection in tilapia culture are the massive mortalities and resistant to various antibiotics like neomycin, oxytetracycline, norfloxacin, nalidixic acid, lincomycin, ampicillin, gentamycin, and tetracycline (Lazado and Zilberg 2018; Abd El Latif et al. 2019). Thus, it is critical to find a different strategy to enhance the general health of fish and reduce antibiotic usage (Ibrahim et al. 2024). As a result, improving disease resistance and enhancing fish’s antioxidant and immune capacity have become necessary in healthful aquaculture (Amer et al. 2022; Abdel Rahman et al. 2023). Currently, aquaculture uses plant supplements as exogenic immune-prophylactics and antioxidants (Thanigaivel et al. 2016; Alagawany et al. 2020; Ahmadifar et al. 2021). The majority of plants and their extracts have quinones, phenolic glycosides, polyphenols, terpenoids, flavonoids, alkaloids, polypeptides, tannins, and polysaccharides substances that are efficient substitutes for antibiotics and synthetical composites (Abdou Said et al. 2021; Nik Mohamad Nek Rahimi et al. 2022). Besides, Medicinal plants are tremendous sources of nutrients for animals, eco-friendly, economical, locally available, and can fend against a wide range of bacteria (Tadese et al. 2022; Nguyen et al. 2023).

Among these medicinal plants, white poplar, Populus alba (P. alba), is a medicinal herb of the Salicaceae (willow) family, which is frequently grown in temperate and subtropical areas (Tawfeek et al. 2019). Its native range extends from the Atlas Mountains in Africa through most of southern and central Europe to Central Asia. It has been introduced into various temperate and humid regions around the world. It grows on moist sites, often next to water, in hot summers and cold to mild winters (Mifsud 2002; Fussi et al. 2010; Costa et al. 2011; Caudullo and De Rigo 2016). Worldwide, 91% of poplars are found in natural forests (Singh and Kumar 2012); nevertheless, introduced forests are small but quickly developing components of poplar forests. There are ∼5 million hectares of poplar plantations worldwide and another 1.5 million hectares in agroforestry systems (Kutsokon et al. 2015). Most (73% of the global population) are farmed in China (FAO 2005). One of the most desirable characteristics of poplar trees is their rapid growth. In the United States, particular studies specify a range of production rates from 5.4 to 30 mg/ha/year, and findings depend on many factors (Riemenschneider et al. 2001; Geyer 2006; Goerndt and Mize 2008).

The efficacy of ethanolic extracts of P. alba leaves against various microorganisms has been established (Nassima et al. 2019). The effective substances from the genus Populus are identified in conventional medication for diverse biological activities involving antioxidant, antiseptic, antiviral, antifungal, and antitumoral (Tebbi and Debbache-Benaida 2022). Incorporating P. alba extract into an agar medium showed the antimycobacterial effect against the growth of three types of mycobacteria due to their content of flavonoids and polyphenols (Haouat et al. 2013). An earlier study showed that chitosan white poplar nanocapsule had an in vitro antibacterial activity against Streptococcus agalactiae (Abdel Rahman et al. 2023). We designated this study based on the detrimental impacts of A. veronii in tilapia culture, the resistance of this strain to various antibiotics, the urgency to reduce antibiotic usage in aquaculture, and the potential therapeutic effect of white poplar extract (WPE). The in vitro antibacterial action of WPE against A. veronii was assessed. In addition, the potential therapeutic effects of WPE on the antioxidant activity, immune status, and blood biochemical parameters of Nile tilapia challenged with A. veronii were evaluated.

Materials and methods

Preparation of WPE

Fresh white poplar leaves were obtained from the Faculty of Agriculture, Benha University, Egypt, and were identified by the classification unit at the Desert Research Centre, Cairo, Egypt. Dehydrated white poplar leaves were ground into a powder with milling; then, 10 g of flour was mixed with 100 mL acidified ethanol (70%) solution [0.1 M HCl (v/v)] to prepare the ethanolic extract according to Zhao et al. (2008).

Determination of the phytochemical constituents of the plant extract

The WPE's qualitative phytochemical components, including its flavonoids, phenols, terpenes, steroids, alkaloids, cardiac glycosides, saponins, and tannins, were evaluated using Trease and Evans’ standard methodology (Trease and Evans 1989).

Gas chromatography-mass spectrometry analysis (GC-MS)

At the Central Laboratories Network, National Research Centre, Cairo, Egypt, a gas chromatograph (GC) (7890B) and mass spectrometer detector (5977 A) were part of the GC-MS system (Agilent Technologies). An HP-5MS column with dimensions of 30 × 0.25 mm internal diameter and 0.25 μm film thickness was installed in the GC. The following temperature program was used during the analysis, with hydrogen serving as the carrier gas at a rate of 1.0 mL/min at a splitless injection volume of 1 µl (temperature: 50 °C for 1 min; rising at 10 °C/min to 300 °C and held for 20 min). The injector and detector were kept at 250 °C. Mass spectra were generated by electron ionisation (EI) at 70 eV with a spectral range of m/z 30–700 and a solvent delay of 9 min. The Quad was 150 °C, but the majority was 230 °C. Identifying numerous chemicals by contrasting the spectrum fragmentation pattern with those seen in the Wiley and NIST Mass Spectral Library data was feasible.

Isolation of the bacterial strain

Aeromonas veronii strain (131TF-ID) was isolated (in the Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Egypt) from sick O. niloticus. The strain was identified using the VITEK^® 2 system (BioM' Erieux Inc., NC, USA) and the universal bacterial primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) according to Reda et al. (2022). The isolate was grown for 24 h at 27 °C on tryptic soy agar, and then one colony was incubated in tryptic soy broth for another 24 h at 27 °C. The pellet from the A. veronii cultured broth was collected and suspended in a sterile phosphate-buffered saline solution after centrifuging for 10 min at 3000 rpm at 4 °C. Aeromonas veronii’s lethal dosage 50 (LD₅₀) was established (Ibrahim et al. 2024). Five groups were created using 100 fish with two replicates (10 fish/replicate; 20 fish/group). The fish were deprived of food for 24 h before the experimental infection. The fish groups were intraperitoneally (IP) injected with bacterial suspension at various concentrations, which were adjusted by the McFarland standard tube. A 0.2 mL of A. veronii (10⁵–10⁸ CFU/fish) was injected into the first through fourth groups, and 0.2 mL of sterile saline was injected into the fifth group. Four days following the injection, the fish deaths were noted. The LD₅₀ was calculated to be 2 × 10⁷ CFU/mL based on the results of the Probit Analysis Programme (version 1.5). A sub-lethal dosage of 0.5 × 10⁷ was determined and used in the treatment study.

In vitro study for determining the antibacterial activity

Disc diffusion assay

The disc diffusion technique was used to determine the zone of inhibition during the antibacterial screening of the extracts (Liu et al. 2016). On Muller Hinton agar plates (Oxoid, England), 100 µL of a bacterial suspension containing 0.5 × 10⁷ CFU/mL (equal to 0.5 McFarland turbidity) was uniformly streaked. The striped plates were placed in the incubator and dried for 20 min at 37 °C. Onto sterile Whatman paper discs (5 mm in diameter) were soaked in 10 µL of WPE solution (1.5 µg/mL). The discs were also dried in an incubator. The cefotaxime antibiotic disc (10 µg, Oxoid, England) (Vila et al. 2002) was used as a positive control, and a disc soaked in ultra-purified water (10 µL) was utilised as a negative control. The dried discs were added to the inoculation plates and incubated for 24 h at 37 °C. The inhibitory zones were then calibrated to the nearest millimetre (mm). The assay was carried out in triplicates.

Minimum inhibitory concentration (MIC)

WPE was subjected to the MIC procedure (Swain et al. 2014). In brief, we prepared serial dilutions (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg/mL) from the extract. The cultures containing 0.5 × 10⁷ CFU/mL of the bacterial isolate were added, and the 96-well plate was incubated, as mentioned before. The MIC level was at which there was no sign of bacterial growth on the plate.

Minimum bactericidal concentration (MBC)

MBC was achieved by sub-culturing from each well onto a nutrient agar plate. For that test strain, the plate with the lowest extract concentration that failed to produce growth on subculture was regarded as MBC (Ibrahim et al. 2023).

Fish

Two hundred sixty O. niloticus (33 ± 1.5 g) were acquired from the Central Laboratory of Aquaculture Research in Abbasa, Egypt. Following CCAC (2005) recommendations, they were examined and stocked as ten fish per glass tank (50 × 40 × 60 cm) with 90 L water volume. The fish were initially held for a 14-day adaptation period, during which they were fed a commercial diet and monitored for disease and mortality. The physiochemical characteristics of water were observed according to APHA (1992). Every day at 9:00 am, the water parameters were measured using an oxygen metre (970 portable DO metre, Jenway, London, UK) for dissolved oxygen (DO) and water temperature (T; °C). A pH metre (Digital Mini-pH Metre, model 55, Fisher Scientific, Denver, CO, USA) was used to measure the pH. Salinity was measured using a refractometer (Erma, Japan). Using the High Range Ammonia Colorimeter—Checker^® HC HI733 (HANNA, Egypt), the ammonia was estimated. The temperature range was 26 ± 1.5 °C, dissolved oxygen was 6.5 ±0.3 mg L⁻¹, and ammonia was 0.02 ± 0.003 mg L⁻¹. Each tank’s water was physically sucked out every other day and replenished with fresh water.

Determination of the therapeutic dose of WPE

For determination of the therapeutic concentration of WPE, one hundred fish were divided into 10 groups and subjected to 10 grading concentrations of WPE (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 mg/L) for 10 days (Table 1). The WPE was dissolved in 0.03% dimethyl sulfoxide (DMSO) before being added to the aquarium water. Daily siphoning of the excretory debris was conducted during the exposure period, and clinical signs and mortalities were recorded. The 0.5–1.5 mg/L range was safe for WPE concentration limits, as it did not result in any abnormal clinical signs or mortality. A concentration of 1.5 mg/L WPE was chosen as the therapeutic dosage.

Table 1. Effect of water exposure to different white poplar extract (WPE) levels on mortality and clinical signs of O. niloticus for 10 days.

Conc., mg/L	Mortality, % (N = 10)	Clinical observations
		Loss of escape reflex	Swimming abnormalities	Skin lesions
0.0	0	−	−	−
0.5	0	−	−	−
1.0	0	−	−	−
1.5	0	−	−	−
2.0	10	−	−	−
2.5	10	−	−	−
3.0	10	−	−	−
3.5	20	+	+	+
4.0	20	+	++	++
4.5	30	+++	+++	+++

(−) No abnormal observations, (+) Mild abnormal observations, (++) Moderate abnormal observations (+++), Sever abnormal observations.

Experimental design and protocol

The experimental design is shown in Figure 1. One hundred sixty fish were assigned into four experimental groups (4 glass tanks/group, 40 fish/group, 10 fish/tank) for ten days. In the first and second groups, 0 and 1.5 mg/L WPE were applied in tank water, respectively, without fish being challenged. In the 3rd and 4th groups, 0 and 1.5 mg/L WPE were applied in tank water, respectively, with fish intraperitoneal inoculated with 0.2 mL of A. veronii (0.5 × 10⁷ CFU). Throughout the experiment, the fish were given a commercial meal three times a day until satisfied. Fish were kept under constant observation for any clinical symptoms or death.

Figure 1. A schematic depicts the research designs (groups, treatments, duration, and laboratory investigations).

Sampling

After the experiment (10 days), fish were starved for 24 h and sedated by a 100 mg/L benzocaine solution (Lugo et al. 2008). Blood samples were taken from the caudal vessels of 12 fish/group into two batches. The first batch was taken using a 1 mL plastic syringe and left at room temperature (23 °C) for 4 h before being centrifuged at 1750 ×g for 10 min to separate the serum. Sera was then stored at −20 °C until use in biochemical and immunological testing. A 1 mL heparinised syringe was used to collect the second batch of blood samples for the phagocytic activity assay. To analyse the hepatic oxidant/antioxidant assays, liver samples (n = 12) from the deceased fish were also taken.

Liver antioxidant indices

The hepatic content of malondialdehyde (MDA) was spectrophotometrically measured in the homogenate using a specific kit (Bio-diagnostic company, Giza, Egypt; Catalog No. MD 25 29), while the catalase activity (CAT) was measured as described by Aebi (1984).

Immunological indices

The kits from Cusabio Co. (Houston, TX, USA; Catalogue no. CSB-E12045Fh) were used to measure the immunoglobulin M (IgM) serum level. Following Ghareghanipoora et al. (2014), with minor changes, we assessed serum lysozyme (LYZ) activity using the lysis of Micrococcus lysodeikticus (Sigma Co., MO, USA). We combined the serum with the M. lysodeikticus solution (0.2 mg/mL in 0.05 M PBS, pH 6.2) at 25 °C for 5 min. We employed a 5010 photometer from the BM Co. in Berlin, Germany, to measure the optical density at 540 nm for 5 min at 1-min intervals using various dilutions of lyophilised chicken egg-white lysozyme (Sigma Co., MO, USA). Following Bryan and Grisham (2007) methodology, nitric oxide (NO) was evaluated. Based on the methodology of Cai et al. (2004), leukocytes’ phagocytic activity (PA%) was assessed. $$\mathit{\text{Phagocytic activity}}\mathrm{\%}=\frac{\mathit{\text{No}}.\mathit{\text{of phagocytic cell phagocytizing bacteria}}}{\mathit{\text{Total no}}.\mathit{\text{of phagocytic cells counted}}}\times 100$$

Serum biochemical indices

The serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using the kits of Biotrend Co., MD, USA (Catalog no. EK12276 and MBS038444, respectively). The creatinine level was determined as described by Fossati et al. (1983). The electrophoretic distribution of serum proteins, including total proteins (TP), albumins, and globulins, was also measured (Badawi 1971). The serum glucose level was assessed following the technique described by Hyvarinen and Nikkila (1962). The serum cortisol level was measured according to Odhiambo et al. (2020).

Statistical analysis

Shapiro-Wilk’s test was used to check the data’s normality. The outcomes of the antioxidant/immune and biochemical indices were examined using a two-way analysis of variance (ANOVA) in SPSS version 18 (SPSS, Chicago, IL, USA). Tukey’s multiple range tests were used to discover differences between means (n = 12/group). The data variation was represented as pooled SEM, and the significance level was established at p < 0.05.

Results

Phytochemical composition of the plant extract

The active phytochemical compounds of WPE are presented in Table 2. WPE had abundant terpenoids and moderate amounts of phenolics and steroids, then low amounts of alkaloids, tannins, flavonoids, saponins, and glycosides.

Table 2. Phytochemical compounds in white poplar extract (WPE).

Constituents	WPE
Phenolics, %	2.83
Terpenoids, %	3.94
Steroids, %	2.35
Saponins, %	0.69
Flavonoid, %	0.21
Alkaloids, %	0.20
Tannins, %	0.09
Glycosides, %	0.06

GC-MS results

The result of GC-MS of WPE is shown in Table 3 and Figure 2. GC-MS analysis detected 17 bioactive compounds in WPE. The major substances are 9,12,15-Octadecatrienoic acid, methyl ester, (Z, Z, Z)-(19.96%), Phytol (16.67%), beta.-Amyrone (14.59%), Methyl tetradecadienoate (10.44%), Hexadecanoic acid, ethyl ester (10.08%), γ-Sitosterol (9.24%), Phosphoric acid (7.63%), Stearic acid (2.53%), β-Sitosterol, TMS derivative (2.44%), Vitamin E (1.65%).

Figure 2. GC-MS chromatogram of white poplar leave extract.

Table 3. Bioactive compounds in white poplar extract (WPE) determined by GC-MS.

Peak	Name	Formula	RT, min	Peak area, %)
1	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)	C19H32O2	29.752	19.96
2	Phytol	C20H40O	29.271	16.67
3	beta.-Amyrone	C30H48O	38.083	14.59
4	Methyl tetradecadienoate	C15H26O2	29.643	10.44
5	Hexadecanoic acid, ethyl ester	C18H36O2	28.127	10.08
6	γ-Sitosterol	C29H50O	38.026	9.24
7	Phosphoric acid, dioctadecyl ester	C36H75O4P	29.809	7.63
8	Stearic acid	C18H36O2	28.030	2.53
9	β-Sitosterol, TMS derivative	C32H58OSi	38.209	2.44
10	Vitamin E	C29H50O2	36.802	1.65
11	Catechol, TMS derivative	C9H14O2Si	17.404	1.13
12	3-Butyn-2-amine, N-methyl	C5H9N	12.357	0.57
13	Pyrocatechol, TMS	C12H22O2Si2	19.836	0.42
14	Dihydroergosterol	C28H46O	29.037	0.5
15	Octadecanoic acid, 2-methyl-, methyl ester	C20H40O2	29.912	0.57
16	Heptacosane	C27H56	33.929	0.76
17	Ethylene glycol diacrylate	C8H10O4	38.329	0.82

In-vitro antibacterial activity of WPE

Figure 3 showed that WPE had an inhibition zone of 22 ± 0.45 mm against A. veronii. Meanwhile, cefotaxime gave a 24 ± 0.33 mm inhibitory zone. The MIC and MBC of WPE against A. veronii were 60 µg/mL.

Figure 3. The in vitro anti-bacterial activity of white poplar extract (WPE) against A. veronii. (A) WPE (1.5 μg/mL). (B) Positive control (cefotaxime, 10 μg/disc). (C) Negative control (ultrapure water).

Clinical signs and survival

The non-infected fish treated with 0 and 1.5 mg/L WPE exhibited normal clinical signs with a 100% survival rate. While the infected fish treated with 0 mg/L WPE displayed decreased swimming activity with a manifestation of skin haemorrhage and fin rot as well as the lowest survival rate (67.5%) (Figure 4). Interestingly, the infected fish treated with 1.5 mg/L WPE retrieved the previous clinical manifestations, only for slight fin rot with enhanced survivability (90%) compared to the infected-nontreated group.

Figure 4. Survivability (%) of O. niloticus in the different experimental groups. WPE: white poplar extract.

Antioxidant-immune status

The effect of WPE application on the antioxidant-immune status of O. niloticus challenged with A. veronii is shown in Table 4. Regardless of WPE application, challenging the fish with A. veronii resulted in detrimental effects on the antioxidant-immune status of fish, indicated by a rise in the oxidative status (increased hepatic MDA level) and reduction in the antioxidant activity (reduced hepatic CAT activity) (p < 0.01). Furthermore, the reduction in the immune status of fish is represented by lowered LYZ, NO, IgM, and PA % (p < 0.01). Regardless of A. veronii challenge, WPE enhanced both antioxidant and immune responses in fish (p < 0.01). The interaction between bacterial challenge and WPE application showed boosted antioxidant activity (p < 0.01) and immune responses (p < 0.05) of fish treated with WPE during the bacterial challenge.

Table 4. Effects of white poplar extract (WPE) application on immunological parameters of O. niloticus challenged with A. veronii.

Groups	MDA, nmol/g	CAT, U/dL	LYZ, ng/ml	NO, mg/dl	IgM, µg/mL	Phagocytosis, %
Non-infected	14.52^c	39.81^a	115.12^b	36.34^b	9.71^b	54.07^b
Non-infected + WPE	14.22^c	41.62^a	126.59^a	39.04^a	19.59^a	66.57^a
Infected	93.85^a	18.74^c	32.21^d	16.34^d	3.34^d	22.68^d
Infected + WPE	45.17^b	28.89^b	42.36^c	26.04^c	5.44^c	35.13^c
SEM	9.82	2.82	12.71	2.20	0.82	5.11
Two-way ANOVA, p-value
A. veronii infection	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
WPE	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01
Interaction	<0.01	0.01	0.01	0.02	0.03	0.01

MDA: malondialdehyde; CAT: catalase; LYZ: lysozyme; NO: nitric oxide; IgM: immunoglobulin M.

Variation in the data was expressed as pooled SEM (n = 12/group). Values in the same column that did not share the same superscripts are significantly different (p < 0.05; two-way ANOVA).

Blood biochemical indices

Regardless of the WPE application, the challenge with A. veronii increased the hepatorenal indices (ALT, AST, and creatinine), cortisol, and glucose with a significant decline in TP and albumins (p < 0.01) with no significant effect on the serum globulin level (p > 0.05) (Table 5). Regardless of A. veronii challenge, WPE application decreased the serum levels of hepatorenal indices, cortisol, and glucose and increased serum total protein and albumin levels (p < 0.01). The interaction between the bacterial infection and WPE application revealed increased ALT levels in infected fish (not treated with WPE) while decreased in fish treated with WPE during the bacterial challenge. The serum creatinine, cortisol, and glucose levels were increased in infected fish (not treated with WPE). In contrast, these levels were decreased in fish treated with WPE during the infection but still higher than in the non-infected fish either treated or not treated with WPE (p < 0.01). The opposite was observed in the serum levels of TP and albumin. The interaction did not affect Serum AST and globulin levels (p > 0.05).

Table 5. Effects of white poplar extract (WPE) application on blood biochemical parameters of O. niloticus challenged with A. veronii.

Groups	ALT, U/L	AST, ng/mL	Creatinine, mg/dL	TP, g/dL	Albumin, g/dL	Globulin, g/dL	Cortisol, ng/mL	Glucose, mg/dL
Non-infected	21.60^c	15.20	0.337^c	3.28^a	1.81^a	1.470	28.30^c	32.70^c
Non-infected + WPE	20.80^c	16.10	0.320^c	3.03^ab	1.79^a	1.230	27.20^c	32.30^c
Infected	61.80^a	40.10	0.880^a	2.18^c	1.19^c	0.997	65.00^a	72.30^a
Infected + WPE	42.30^b	32.50	0.613^b	2.56^b	1.34^b	1.220	52.00^b	61.40^b
SEM	5.09	17.60	0.069	0.136	0.082	0.064	4.85	5.32
Two-way ANOVA, p-value
A. veronii infection	<0.01	<0.01	<0.01	<0.01	<0.01	0.062	<0.01	<0.01
WPE	<0.01	0.196	<0.01	0.574	<0.01	0.955	<0.01	<0.01
Interaction	<0.01	0.112	<0.01	0.022	<0.01	0.078	<0.01	<0.01

ALT: alanine aminotransferase; AST: aspartate aminotransferase; TP: total proteins.

Variation in the data was expressed as pooled SEM (n = 12/group). Values in the same column that did not share the same superscripts are significantly different (p < 0.05; two-way ANOVA).

Discussion

Providing safe substitutes to antibiotics for treating bacterial pathogens in aquaculture is a must for reducing the antibiotic resistance problem. Phytotherapeutics are effective and eco-friendly ways to treat fish diseases without compromising the sustainability of the environment. Herbal extracts can be used as impressive natural alternatives to antibiotics or chemical drugs for treating fish illness, reducing drug resistance and residual accumulation problems in fish and consumers. The current study proposed a water additive (WPE) for controlling the A. veronii challenge in Nile tilapia. The group infected with A. veronii displayed decreased swimming activity, diseased signs (fin rot and skin haemorrhage), and the lowest survivability (67.5%). These outcomes might be due to the bacteria’s virulence factors (phospholipase, adhesin, and aerolysin), which possess cytotoxic and haemolytic activities; these virulence factors are held responsible for the development of such clinical manifestation (Li et al. 2020). Treatment of the infected fish with WPE retrieved the previous clinical manifestation, only for slight fin rot and improved survivability (90%). These results could be due to WPE antibacterial activity, which was approved by the in-vitro investigations. WPE had an inhibitory zone against A. veronii of 22 ± 0.45, with MIC and MBC of 60 µg/mL. The antibacterial activity of WPE in the current study could related to its principal constituent, ɑ-linolenic acid (9,12,15-Octadecatrienoic acid, methyl ester, (Z, Z, Z), which was previously documented to have antibacterial activity against Bacillus subtilis (Kusumah et al. 2020) and Mycobacterium tuberculosis (Masoko et al. 2016). Phytol, detected in our GC/MS analysis, had antibacterial activity against Pseudomonas aeruginosa (Lee et al. 2016). Previous studies showed that WPE displayed an in vitro antibacterial activity against Mycobacterium tuberculosis (Haouat et al. 2013), Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis (Nassima et al. 2019). A previous study carried out by Abo El-Fadl et al. (2022) documented that WPE has various active constituents which have antimicrobial, anti-inflammatory, antioxidant, and immunomodulatory effects.

Extreme reactive oxygen species (ROS) production can damage biomolecules, including lipids, nucleic acid, and proteins, which oxidate lipids and protein carbons (Zheng et al. 2019). Aeromonas veronii infection significantly decreased the hepatic CAT. It increased the hepatic MDA (lipid peroxidation marker) level. In this line, Elgendy et al. (2022) observed that Nile tilapia, challenged with A. veronii showed a reduction in the antioxidants and an increase in MDA level. Herein, increased MDA levels and lowered CAT activity could be attributed to bacterial toxins’ ROS overproduction in the cell membrane, resulting in such oxidative damage (Bandeira Junior and Baldisserotto 2021). Treating the infected fish with WPE ameliorated the antioxidant activity compared to the infected-nontreated fish. The enhanced antioxidant status of infected fish by WPE treatment could be related to its primary compound [ɑ-linolenic acid (19.96%), phytol (16.67%), and vitamin E (1.65%)]. ɑ-linolenic acid has antioxidant potential by improving the antioxidant enzyme (CAT) and reducing the MDA level (Yuan et al. 2022). Also, phytol was reported to have antioxidant properties (Santos et al. 2013; Okpala et al. 2022). Vitamin E is a non-enzymatic antioxidant molecule with antioxidant properties in spotted seabass (Lateolabrax maculatus) (Li et al. 2023). Also, WPE contains a mixture of flavonoids and phenolic compounds that can resist oxidation, eliminate free radicals, and enhance immune function (Tian et al. 2019). Many medicinal plants have a strong free radicals scavenging ability because their structure contains multiple phenolic hydroxyl groups (hydrogen donors and singlet oxygen quenchers) and indirectly raises the stress resistance ability (Hoseinifar et al. 2017; Alagawany et al. 2020). Phenols and flavonoids can scavenge most oxidative fragments and other free radicals associated with many diseases (Mbokane and Moyo 2018).

The fish’s antioxidant defense system strongly correlates to the immune system and health condition (Zhang et al. 2020). LYZ is an immune protein in mucus, Plasma, and lymphoid tissue (Saurabh and Sahoo 2008). LYZ has a role in antibacterial activity, among other defensive processes (Maqsood et al. 2010). Nitric oxide is involved in the immune function of vertebrates and invertebrates (Sharma et al. 2010). Nitric oxide safeguards the host from bacterial or viral illnesses (Srivastava and Pandey 2015). IgM, the predominant immunoglobulin in teleosts, can immediately protect fish against illnesses (Magnadottir 2010). In addition, Blood protein levels, notably globulin, are assumed to reflect the fish’s improved nutritional condition and immunological response (Sahoo et al. 2021). The current study showed that A. veronii infection significantly decreased the immunological parameters (LYZ, NO, IgM, and PA %) and decreased TP, albumin, and globulins. The decreased immunological biomarker in the current experiment caused by A. veronii infection was similar to those obtained by Reyes-Becerril et al. (2015), who found comparable outcomes in Pacific red snapper (Lutjanus peru) challenged with A. veronii. Treatment of the A. veronii-challenged fish with WPE improved the immune status and protein profile significantly compared to the infected-nontreated fish. The immunostimulant properties of WPE could be related to its active constituent; Phytol had immunostimulant activity by improving the serum level of LYZ and IgM in common carp (Cyprinus carpio) (Hoseini et al. 2021). Also, WPE has phytochemical constituents like phenols, terpenes, tannins, and alkaloids. Phenolics could inhibit pathogenic bacterial growth (Citarasu 2010). Terpenes may also have immune-modulating effects by promoting T and B cell activation (Harikrishnan et al. 2011b). Tannins had an immune-stimulatory impact on Beluga sturgeon (Huso huso) by boosting LYZ levels (Safari et al. 2020). In the Blunt snout bream (M. amblycephala), alkaloids, another biological component of WPE, were found to have an immune-stimulatory impact by raising IgM levels and boosting resistance to Aeromonas hydrophila (Ye et al. 2019).

In the current study, A. veronii infection significantly increased the serum levels of hepatorenal indices. Elgendy et al. (2022) achieved similar outcomes. The elevated hepatorenal indices caused by A. veronii infection could be due to damaged hepatocytes and renal cells by A. veronii virulence components, increasing these indicators in the blood (Abd El Latif et al. 2019). Treatment of the infected fish with WPE ameliorated these indices compared to the infected-non-treated one. These results may be attributed to the protecting abilities of WPE. WPE may enhance cell membrane stability and protect tissues from toxic damage caused by free radicals through the antioxidant properties of its active substances, resulting in decreased levels of ALT and AST.

Cortisol and glucose are considered stress indicators in fish (Ahmed et al. 2022; Ibrahim et al. 2023). Aeromonas veronii infection elevated the serum cortisol and glucose. Water exposure of the infected fish to WPE reduced these stress biomarkers. These indicated the anti-stress and anti-inflammatory properties of WPE due to its active constituents (flavonoids, terpenes, and phenolics). Terpenes and phenols have anti-inflammatory activities (Aggarwal et al. 2006). Also, flavonoids decreased the level of cortisol and glucose in rainbow trout (Gesto et al. 2008).

Conclusions

From the obtained results, we can conclude that WPE has in vitro antibacterial activity (22 ± 0.45 mm inhibition zone, with MIC and MBC of 60 µg/mL) against A. veronii). In addition, WPE (1.5 mg/L) can be used as an antibacterial substance in fish farming to alleviate the detrimental impacts of A. veronii infection by enhancing the antioxidant/immune status of the infected fish indicated by the increase in the serum levels of CAT, LYZ, NO, IgM, and PA % and reducing MDA level. WPE application could reduce the harmful effects of A. veronii infection on the hepato-renal function and reduce stress caused by the infection. These outcomes could help reduce antibiotic usage in fish farms and alleviate Nile tilapia’s health and immune responses during A. veronii challenge. Additional studies are required to examine the effect of WPE as an affordable product for farmers at the fish farm level.

Ethical approval

The experimental protocol received ethical approval from Zagazig University’s Institutional Animal Care and Use Committee (Approval No. ZU-IACUC/2/F/136/2021). All animal experiments were carried out in accordance with the instructions outlined in ‘The Guide for the Care and Use of Laboratory Animals in Scientific Investigations’, and the study followed the relevant institutional standards. All animal experiments were carried out in accordance with the ARRIVE guidelines.

Acknowledgements

This work was supported by the Researches Supporting Project (RSP2024R36), King Saud University, Riyadh, Saudi Arabia. The authors thank Professor Rasha M. Reda for her kind help and providing the A. veronii strain. The authors thank the Aquatic Animal Medicine Department, Faculty of Veterinary Medicine, Zagazig University, for their kind help during the experimental procedures.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets generated or analysed during the current study are not publicly available but are available upon reasonable request from the corresponding author.