Modulatory effects of fenugreek seeds powder on the histopathology, oxidative status, and immune related gene expression in Nile tilapia (Oreochromis niloticus) infected with Aeromonas hydrophila

Aquaculture 515 (2020) 734589

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Modulatory effects of fenugreek seeds powder on the histopathology, oxidative status, and immune related gene expression in Nile tilapia (Oreochromis niloticus) infected with Aeromonas hydrophila

T

Eman M. Moustafaa, Mahmoud A.O. Dawoodb,∗, Doaa H. Assarc, Amira A. Omara, Zizy I. Elbialyd, Foad A. Farrage, Mustafa Shukryf, Mohamed M. Zayedg a

Department of Fish Diseases and Management, Faculty of Veterinary Medicine Kafrelsheikh University, Egypt Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, Kafrelsheikh, Egypt Department of Clinical Pathology, Faulty of Veterinary Medicine, Kafrelsheikh University, Egypt d Department of Fish Processing and Biotechnology, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Egypt e Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt f Department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt g Department of Aquaculture, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Kafrelsheikh, Egypt b c

ARTICLE INFO

ABSTRACT

Keywords: Fenugreek Aeromonas hydrophila Nile tilapia Histopathology Immune related gene expression

The present trial investigated the effects of dietary fenugreek on the histopathology, oxidative status, and immune-related gene expression in Nile tilapia (Oreochromis niloticus) (15 ± 0.5 g) infected with Aeromonas hydrophila. Fish were fed two diets with or without fenugreek supplementation at 3%. The trial was lasted for 8 weeks then fish were divided into four groups; the first and fourth groups were intra-peritoneally injected with phosphate buffer saline (PBS) while the second and third groups were challenged with sublethal dose of A. hydrophila. At 6 h, 24 h and 1-week post challenge, several serum immune and immune-related gene expressions and antioxidant defense parameters as well as the histopathological alterations were evaluated. The results indicated that administration of fenugreek supplemented diets could significantly increase the growth performance parameters and decreased the feed conversion ratio as compared to the non-supplemented groups (P < 0.05). Furthermore, fish fed fenugreek supplemented diets showed improved immune (lysozyme, immunoglobulin, and respiratory burst activity) and antioxidant parameters (superoxide dismutase and glutathione peroxidase and malondialdehyde) in 6 h, 24 h and 1-week post challenge (P < 0.05). Additionally, fenugreek upregulated the immune-related gene expressions in liver and kidney (IL-1β and TNF-α) and reduced the blood aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicating the protective role for the hepatic tissue (P < 0.05). The histopathological images of A. hydrophila infected groups revealed damage in intestine, spleen, hepatopancreas and kidney which exaggerated with time, but the infected groups fed on fenugreek diet showed significant alleviation of damage of intestine, hepatopancreas, spleen and kidney. The results of challenge test showed that administration of fenugreek supplemented diets significantly increased tilapia resistance (P < 0.05); the highest survival rate, was observed in the third group. Fish fed 3% fenugreek supplemented diets exhibited increased immune response and antioxidant capacity compared other groups (P < 0.05). The present results confirmed the beneficial effects of fenugreek as a natural alternative immunostimulant for Nile tilapia aquaculture.

1. Introduction Nowadays, several researchers are interested in finding suitable dietary additives to apply in aquaculture instead of synthetic antibiotics owing its extreme down-regulation of metabolic enzymes, muscular development and resurgence of virulent pathogens in aquatic biota ∗

(Amin et al., 2019; Dawood et al., 2018, 2019b; Van Doan et al., 2019a). For these factors, several countries completely prohibited the application of chemical drugs in aquaculture practices (Abdelkhalek et al., 2017; Adel et al., 2017). Usually, medicinal herbs are considered as potential immune enhancers and antioxidants when applied in the land-based captive, hatchery and farmed animals (Adel et al., 2016;

Corresponding author. E-mail addresses: [email protected], [email protected] (M.A.O. Dawood).

https://doi.org/10.1016/j.aquaculture.2019.734589 Received 29 August 2019; Received in revised form 4 October 2019; Accepted 8 October 2019 Available online 09 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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Ahmadifar et al., 2019a, 2019b; El-Deep et al., 2019; Saleh et al., 2019; Van Doan et al., 2019b, 2019c). These additives could augment the growth of beneficial microbial colonies in the digestive tract which lead to enhance feed intake and weight gain of various aquatic species (Safari et al., 2017). Enhanced resistance against infectious diseases of aquatic species following the administration of different medicinal plants was also shown previously (Van Doan et al., 2019b, 2019c; Yin et al., 2009). Fenugreek (Trigonella foenum graecum) have been long considered to be health-promoting, as far back as ancient times (Kaviarasan et al., 2007; Paul and Pal, 2014). The seeds contain many bioactive compounds including, polyphenols, nitrogenous compounds, vitamins, inorganic elements, carbohydrates, lipids, proteins, free sugars, and mucilage (Mukthamba and Srinivasan, 2016). Previous studies have demonstrated the beneficial impacts of fenugreek extract on antidiabetic, antiplasmodic, hypolipidemic, antibacterial, anthelmintic, anti-inflammatory, and analgesic. It also has antioxidant, anticarcinogenic, antiulcer, antifertility, immunomodulatory effects and acts as an enzymatic pathway modifier (Olaiya and Soetan, 2014). In aquaculture sector, fenugreek seeds powder effectively enhanced the growth performance, immunity and wellbeing of gilthead seabream (Sparus aurata L.) (Awad et al., 2015; Bahi et al., 2017; Guardiola et al., 2017a, 2017b, 2018a, 2018b), common carp (Cyprinus carpio L.) (Roohi et al., 2017), and striped catfish (Pangasius hypophthalmus) (Mehboob et al., 2017). On the other hand, no studies have been conducted to explore the positive effects of fenugreek on improving the immune response and oxidative status of Nile tilapia. Nile tilapia (Oreochromis niloticus), remains one of the most commonly cultured fish species worldwide, due to their easy breeding, tolerance to varied environments and diseases, fast growth, and high market demand (Dawood et al., 2019a, 2019c; Elumalai et al., 2019). However, it faces great challenges due to the infection of Streptococcus spp., Vibrio spp., Aeromonas hydrophila and Flavobacterium spp. Among the pathogen is A. hydrophila, one of the more severe forms of bacteria, which threatens the health of fish by causing Motile Aeromonas Septicemia, affecting cultured freshwater fishes, including tilapia (Cipriano et al., 1984; Yin et al., 2009; Zahran et al., 2018). A. hydrophila has developed in the most damaging impediment to the expansion of the tilapia industry worldwide. In the present work, the main target was to evaluate the effect fenugreek in reducing the damaging effect of A. hydrophila on the vital immune organ tissues of Nile tilapia in concurrent with evaluation to its protective and immunomodulatory effects as well as the antioxidative response.

Table 1 Basal diet and proximate chemical composition (on dry matter basis). Ingredient

%

Chemical composition

%

Fish meal Soybean meal Wheat bran Yellow corn Rice bran Fish oil Dicalcium phosphate Vitamins and minerals mixturea

10 44.4 10 18.6 10 5 1 1

Dry matter Crude protein Ether extract Total ash Gross energy (kcal/100 g)b

92.8 30.9 7.1 7.2 446

a Vitamin mixture (except vitamin E, mg kg−1 premix): vitamin A (3300 IU), vitamin D3 (410 IU), vitamin B1 (133 mg), vitamin B2 (580 mg), vitamin B6 (410 mg), vitamin B12 (50 mg), biotin (9330 mg), colin chloride (4000 mg), vitamin C (2660 mg), inositol (330 mg), para-amino benzoic acid (9330 mg), niacin (26.60 mg), pantothenic acid (2000 mg), manganese (325 mg), iron (200 mg), copper (25 mg), iodine, cobalt (5 mg). b Gross energy was calculated as 5.65, 9.45, and 4.11 kcaL/g for protein, lipid, and carbohydrates, respectively.

2017a, 2017b, 2018a, 2018b). Two experimental diets were prepared to include fenugreek at 0 or 3%. Basal diet formulations were prepared as indicated by Dawood et al., 2019a, 2019c (Table 1). The first group (6 aquaria) fed the basal diet without fenugreek supplementation, while, the second group (6 aquaria) were fed fenugreek -supplemented diet at 3%. Fish fed the test diets twice a day (8:00 and 16:00 h) up to the satiation level for 8 weeks. Water quality items were: DO 6.5 ± 0.5 mg/l, pH 7.1 ± 0.8, EC 219 ± 2 μ mho/cm, temperature 23 ± 2 °C and day and night photoperiod 12:12 h during the trial period. During the trial, experimental fish were checked regularly, and dead fish were removed to eliminate the water deterioration. After 8 weeks the all fish were weighed to calculate the growth performance parameters using the following equations: Weight gain (%) = (W1 – W2) × 100/W1. Specific growth rate (% per day) = {(Ln (W2) –Ln (W1)) / duration (8 weeks)} × 100 Feed conversion ratio = dry feed intake (g) / live weight gain (g). Where W1 and W2 are initial and final body weight (g) of fish. 2.3. Challenge with A. hydrophila A. hydrophila strain was personally provided by the Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt. A. hydrophila was prepared by culturing on Tryptic Soy Agar (TSA) at 37 °C for 24 h with constant shaking (250 rpm). The bacterial count was determined by standard dilution and plating methods, and then the bacterial fluid was diluted to the density of 1.3 × 108 CFU/ml. The LD50 (lethal dose, the dose which kill 50% of the injected fish) was calculated before final injection challenge according to Reed and Muench (1938). The experimental challenge was carried out by intra-peritoneal injection with 0.2 ml fresh culture suspension of A. hydrophila according to Li et al. (2011). Fish were divided into four groups as presented in Table 2. The first

2. Materials and methods 2.1. Ethical statement All the methods, animal care and experimental protocols used in the present study followed relevant guidelines and regulations of Kafrelsheikh University. 2.2. Fish, diet and experimental design Fish used in the trial were obtained from a local fish farm (Kafrelsheikh, Egypt) and transported to the Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafrelsheikh University. Fish then acclimated for trial conditions and fed ad libtum for 14 days before the trial. Then, fish of similar sizes (15 ± 0.5 g) were randomly stocked into 12 well prepared glass aquaria (40 × 60 × 70 cm) at a density of 20 fish per aquarium. Fenugreek (T. foenum-graecum) seeds, obtained from a local market (Kafrelsheikh, Egypt), were crushed. After reviewing the dosages used in previous reports, we performed dosage tests fenugreek at 3% as the optimal dosage (Awad et al., 2015; Bahi et al., 2017; Guardiola et al.,

Table 2 Experimental design for challenge test (n = 3).

2

Group number

Challenge

Fenugreek

Group1 Group2 Group3 Group4

× ✓ ✓ ×

× × ✓ ✓

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and fourth groups were intra-peritoneally injected with 0.2 ml/fish of Phosphate Buffer Saline (PBS) (3 aquaria each). The second and third groups were intra-peritoneally injected with 0.2 ml/fish of 1.3 × 108 CFU/ml of A. hydrophila strain (3 aquaria each) (Table 2). Mortalities were recorded daily for 15 days and freshly dead fishes were moved for further examination and histopathological studies.

Table 3 Primers used for qRT-PCR analysis.

2.4. Blood biochemical, oxidative and immunological parameters

Gene

Primer sequence

NCBI Gene bank accession number

Reference

TNF-α

F:5′GGAAGCAGCTCCACTCTGATGA-3′ R: 5′-CACAGCGTGTCTCCTTCGTTCA-3′ F: 5′-CATCGCCTACGGTCTGGACAA-3′ R: 5′-TGCCGTCTTCAATGGTCAGGAT-3′ F: 5′-CAAGGATGACGACAAGCCAACC-3′

JF957373.1

Qiang et al. (2016) Qiang et al. (2016) Qiang et al. (2016)

HSP70 IL-1β

At the end of the trial, all fish were anesthetized using 150 mg/l MS222 (Argent Laboratories, Redmond, Washington). Blood samples were collected from the caudal blood vessels of 3 fish per aquaria using a syringe without anticoagulant, and serum was separated by centrifuging the clotted blood 3000 rpm/15 min at 4 °C and stored at −20 °C. Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined calorimetrically at the wavelength 540 nm (Reitman and Frankel, 1957). Antioxidant enzyme activity was evaluated using diagnostic reagent kits as directed by the manufacturer (MyBioSource Inc., San Diego, California, USA). The amount of malondialdehyde (MDA) was evaluated using thiobarbituric acid (Ohkawa et al., 1979). Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were evaluated spectrophotometrically using the techniques outlined by (McCord and Fridovich, 1969), (Aebi, 1984), and (Paglia and Valentine, 1967). The lysozyme activity of sera was assayed according to the method described by Demers and Bayne (1997). The lysozyme substrate was 75 mg/ml Micrococcus lysodeikticus lyophilized cells, suspended in 0.1 M sodium phosphate/citric acid buffer; pH 5.8. A 25 μl of the undiluted serum samples were placed into the 96- well micro plate, in triplicates. A 175 μl of the substrate solution was then added to each micro titer plate well and kept at 25 °C; thereafter, rapidly mixed, the changes in turbidity was measured every 30 s for 5 min at the wavelength 450 nm using the micro plate ELISA reader. The unit of lysozyme present in serum (μg/ml) was obtained by matching with the standard curve made with lyophilized hen egg white lysozyme. Following the manufacturer's procedure, the serum immunoglobulin (IgM) level was evaluated using the ELISA kit particular to fish IgM (Cusabio and Cusab, USA). The nitro-blue-tetrazolium (NBT) assay according to Secombes (1990) quantified respiratory burst activity of the entire blood. The microplate reader (Optica, Mikura Ltd, UK) was used to measure the NBT decrease at 630 nm.

β-actin

R: 5′-AGCGGACAGACATGAGAGTGC-3′ F:5′-CCACACAGTGCCCATCTACGA-3′ R: 5′-CCACGCTCTGTCAGGATCTTCA- 3′

FJ207463.1 XM_ 003460625.2 EU887951.1

Qiang et al. (2014)

Internal reference gene (β-actin, house-keeping gene), heat shock protein 70 (HSP70), tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β).

drop (Uv–Vis spectrophotometer Q5000/Quawell, USA). Two μg of RNA sample was reverse transcribed using SensiFAST™ cDNA synthesis kit (Bioline, United Kingdom) following the manufacturer's manual. Gene expression profiling was performed in a Stratagene MX3000P real-time PCR using gene-specific primer sequences for heat shock protein 70 (HSP70), interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α) genes (Table 3). Real-time PCR amplifications using SensiFast SYBR Lo-Rox kit (Bioline) were performed in 20 μL reaction mixtures that contained 2 μL of cDNA, the gene-specific primers (0.5 μM each), and 10 μL of SYBR. The thermal cycling conditions were, initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Melting curve analyses were performed to validate the specificity of the PCR products beginning at 65 °C and ending at 95 °C, with incremental increases of 0.5 °C every 5s. All genes were tested in duplicates. CT values for each sample were determined and incorporated in “fold change” (2−ΔΔCT), calculation based on the Livak and Schmittgen (2001), and mRNA expressions for each sample were normalized against β-actin as a house keeping gene. 2.7. Statistical analysis Two-way ANOVA was employed to test the effects of dietary fenugreek and challenge, as well as their interactions. Once interaction between the two factors is found, all the four groups were tested by oneway ANOVA to test all level of the two factors together. The data from each group were compared to control group and while differences within the same group and among the different sampling periods for each parameter using Tukey-Kramer. All statistical differences were assessed by one-way ANOVA tests (SPSS version 22, SPSS Inc., Il, USA) with Duncan post-hoc test where differences in experimental groups occurred. The level of significance was accepted at P < 0.05. All data are presented as means ± standard error (SE).

2.5. Histopathology study Two fishes were sampled randomly from control group and five fishes were sampled randomly from the other groups at 6 h, 24 h and 7 days post-challenge with A. hydrophila for histopathological examination. After deep anesthesia using 40% ethyl alcohol, the abdomen was dissected and tissue sample from anterior part of small intestine, liver, spleen and posterior part of kidney were fixed in 10% neutral buffered formalin for at least 24 h. The tissue samples were dehydrated by using ascending concentrations of ethanol (70–100%), cleared in xylene, and embedded in paraffin wax. tissue sections of 5 μm thickness were cut on a microtome (Leica RM 2125), then stained with hematoxylin and eosin (H&E) and examined with a light microscope (Leica DM 5000).

3. Results 3.1. Growth performance After 8 weeks of feeding tilapia with or without fenugreek, fish fed fenugreek showed significantly higher final body weight, weight gain and specific growth rate with decreased feed conversion ratio than the non-supplemented groups (Group1 and Group2) (P < 0.05) (Table 4).

2.6. Total RNA extraction, cDNA synthesis and real-time quantitative PCR assay

3.2. Hepatic function of Nile tilapia

To evaluate gene expression levels liver tissue samples were collected from 9 fish per treatment in liquid nitrogen and stored at −80 °C until analysis. Total RNA was extracted from 50 mg using Trizol (iNtRON Biotechnology) according to the manufacturer's instructions. The integrity of RNA was confirmed by ethidium bromide stained 2% agrose gel electrophoresis. RNA concentration was measured by Nano

Infection with A. hydrophila (Group2) resulted in increased ALT and AST values in fish on 6 h, 24 h and 1-week post challenge significantly compared with that of the negative control (Group1) (P < 0.05) (Table 5). However, supplementation of fenugreek resulted in insignificant changes in ALT and AST values in fish infected (Group3) or 3

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Table 4 Growth performance of Nile tilapia fed diets with or without fenugreek for 8 weeks.

Initial body weight (g) Final body weight (g) Weight gain (%) Specific growth rate (% per day) Feed conversion ratio

Group1

Group2

Group3

Group4

15.02 ± 0.09 44.27 ± 0.55a 194.80 ± 3.96a 1.80 ± 0.02a 1.60 ± 0.12b

14.92 ± 0.10 44.73 ± 0.70a 199.97 ± 6.44a 1.83 ± 0.04a 1.51 ± 0.09b

15.13 ± 0.16 59.12 ± 0.53b 290.65 ± 1.02b 2.27 ± 0.23b 1.20 ± 0.04a

15.05 ± 0.25 60.63 ± 0.28b 303.15 ± 8.37b 2.32 ± 0.03b 1.27 ± 0.01a

*Values expressed as means ± SE (n = 6). Different superscript letters indicate significant differences between groups (P < 0.05). Table 5 ALT and AST of Nile tilapia after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek. Group1

AST (uL/mL) ALT (uL/mL)

6h 24 h 1 week 6h 24 h 1 week

Group2

22 ± 2.1A 21.4 ± 2.4A 21.4 ± 3.1A 4.1 ± 1A 4.2 ± 0.78A 4.01 ± 0.7A

Group3

57.5 ± 20.1B 60.1 ± 14.4B 59.4 ± 4.7B 10.2 ± 2B 9.78 ± 2B 10.2 ± 2.5B

Group4

44 ± 2.1AB 40.1 ± 3.1AB 39.4 ± 2.4AB 8.1 ± 1AB 8.4 ± 1.5AB 8.5 ± 2AB

Two way anova (P value)

30 ± 0.67AB 29.4 ± 4.5AB 29.6 ± 3.2AB 6.2 ± 0.12AB 5.4 ± 0.4AB 6 ± 0.7AB

Fenugreek

Challenge

Fenugreek * challenge

0.668 0.001 0.001 0.836 0.675 0.715

0.001 0.001 0.001 0.001 0.001 0.001

0.001 0.001 0.001 0.001 0.001 0.001

*Values expressed as means ± SE (n = 3). Capital superscript letters indicate significant differences between groups (P < 0.05). Table 6 Oxidative status of Nile tilapia after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek. Group1

SOD (u/ml) GPx (u/ml) MDA (nmol/ml)

6h 24 h 1 week 6h 24 h 1 week 6h 24 h 1 week

Group2

148.2 ± 6.2B 148.4 ± 4.1B 147.5 ± 2.4B 1750 ± 15.2BCb 1550 ± 14.5BCa 1620 ± 12.4BCab 41.1 ± 2.8A 38.9 ± 3.3A 37.45 ± 2.6A

Group3

114.4 ± 3.5A 111.4 ± 4.8A 113.4 ± 4.6A 1100 ± 20.2Ab 989 ± 22.4Aab 824 ± 12.7Aa 55.2 ± 3.9Ba 64.7 ± 4.8Bb 64.4 ± 4.7Bb

Group4

132.5 ± 5.4B 139.5 ± 4.8B 139.4 ± 2.4B 1452.4 ± 20.1B 1450.2 ± 2.1B 1489 ± 4.5B 46.4 ± 2.8ABb 42.5 ± 3.9Aab 40.1 ± 2.4Aa

Two way anova (P value)

147.2 ± 4.5B 200 ± 5.2C 195.4 ± 4.6C 1950.4 ± 24.1C 1895.2 ± 14.6C 1912 ± 14.45C 42.8 ± 4.1Ab 40.1 ± 6.0Aab 37.45 ± 4.7Aa

Fenugreek

Challenge

Fenugreek * challenge

0.001 0.001 0.001 0.001 0.286 0.001 0.075 0.001 0.001

0.001 0.001 0.001 0.001 0.358 0.001 0.040 0.001 0.001

0.001 0.001 0.001 0.001 0.272 0.001 0.007 0.001 0.001

*Values expressed as means ± SE (n = 3). Capital superscript letters indicate significant differences between groups, while small letters refer to differences within the same group and among the different sampling periods for each parameter (P < 0.05). Table 7 Blood immune responses of Nile tilapia after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek. Group1

Lysozyme (u/ml) IgM (mg/dl) NBT (OD 630)

6h 24 h 1 week 6h 24 h 1 week 6h 24 h 1 week

Group2

Ac

99.2 ± 1.5 95.8 ± 2.4Bb 90.45 ± 4.6Aa 3.4 ± 0.4ABa 4.1 ± 0.3Bb 4.9 ± 0.5Cc 0.2 ± 0.01Ba 0.21 ± 0.02Bab 0.25 ± 0.01Bb

Group3

Ab

105.3 ± 2.8 85.5 ± 1.9Aa 90.4 ± 4.7Aab 1.6 ± 0.2A 1.3 ± 0.6A 1.4 ± 0.7A 0.17 ± 0.4Ab 0.15 ± 0.5Aab 0.12 ± 0.1Aa

Group4

Bb

115.5 ± 2.5 100.1 ± 2.2Bab 98.7 ± 5.4Ba 3.2 ± 0.8B 3.6 ± 0.6AB 3.7 ± 0.7AB 0.21 ± 0.04Ba 0.24 ± 0.02Bab 0.29 ± 0.1Bb

Two way anova (P value)

Ac

100 ± 2.5 99.2 ± 1.8Bb 96.4 ± 4.6Ba 5.1 ± 0.4C 5.3 ± 0.6C 5.0 ± 0.7D 0.24 ± 0.01Ba 0.35 ± 0.04Cb 0.29 ± 0.02ABab

Fenugreek

Challenge

Fenugreek * challenge

0.075 0.001 0.001 0.001 0.001 0.001 0.082 0.001 0.001

0.040 0.001 0.001 .0339 0.013 0.001 0.686 0.076 0.001

0.007 0.001 0.001 0.001 0.001 0.001 0.686 0.001 0.001

*Values expressed as means ± SE (n = 3). Capital superscript letters indicate significant differences between groups, while small letters refer to differences within the same group and among the different sampling periods for each parameter (P < 0.05).

non-infected (Group4) with A. hydrophila after 6 h, 24 h and 1 week (P > 0.05). Significant interaction effects of both fenugreek supplementation and A. hydrophila infection were observed in tilapia after 6 h, 24 h and 1 week (P < 0.05) (Table 5).

level significantly compared with that of the other groups on 6 h, 24 h and 1-week post challenge (P < 0.05) (Table 6). Interestingly, after 24 h and 1-week post challenge, SOD showed increased levels in fish fed fenugreek (Group4) significantly compared with the other groups (P < 0.05) (Table 6). Significant interaction effects of both fenugreek supplementation and A. hydrophila infection were also observed in tilapia after 6 h, 24 h and 1 week (P < 0.05) (Table 6). Group2 showed significantly reduced GPX levels, while

3.3. Oxidative status Tilapia infected with A. hydrophila (Group2) showed reduced SOD 4

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Fig. 1. Relative expression of liver and kidney HSP70 of Nile tilapia after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek for 8 weeks. Values are expressed as mean ± SE from triplicate groups. Bars with capital superscript letters indicate significant differences between groups, while small letters refer to differences within the same group and among the different sampling periods for each parameter (P < 0.05).

Fig. 2. Relative expression of liver and kidney IL-1β of Nile tilapia after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek for 8 weeks. Values are expressed as mean ± SE from triplicate groups. Bars with capital superscript letters indicate significant differences between groups, while small letters refer to differences within the same group and among the different sampling periods for each parameter (P < 0.05).

supplementation with fenugreek (Group4) significantly showed increased levels compared with those infected with A. hydrophila on 6 h, 24 h and 1-week post challenge (P < 0.05) (Table 6). Fish infected with A. hydrophila and fed fenugreek showed higher GPX than those infected without fenugreek feeding on 6 h, 24 h and 1-week post challenge (P < 0.05) (Table 6). Within the same group the GPx level was decreased on 6 h, 24 h and 1 week following sampling. Significant interaction effects of both fenugreek supplementation and A. hydrophila infection were also observed in tilapia after 6 h and 1 week (P < 0.05) (Table 6). Group2 showed significantly increased MDA levels compared with those in the other groups on 6 h, 24 h and 1-week post challenge (P < 0.05) (Table 6). Within Group2, MDA level was increased on 6 h, 24 h and 1 week following sampling. Significant interaction effects of both fenugreek supplementation and A. hydrophila infection were also observed in tilapia after 6 h, 24 h and 1 week (P < 0.05) (Table 6).

(P < 0.05) (Table 7). Within all groups, lysozyme activity was decreased on 6 h, 24 h and 1 week following sampling. Similarly, the lowest IgM was shown in Group2, while the highest value was presented in Group4 on 6 h, 24 h and 1-week post challenge (P > 0.05) (Table 7). Tilapia infected with A. hydrophila (Group2) showed reduced NBT level significantly compared with that of the other groups on 6 h and 1week post challenge (P < 0.05) (Table 7). The lowest NBT was shown in Group2, while the highest value was presented in Group4 on 24 h post challenge (P > 0.05) (Table 7). In all cases infected fish fed diets supplemented with fenugreek (Group3) showed improved IgM, NBT and lysozyme activity than those fed diets without fenugreek (Group2). Significant interaction effects of both fenugreek supplementation and A. hydrophila infection were also observed on IgM, NBT and lysozyme activity of tilapia after 6 h, 24 h and 1 week (P < 0.05) (Table 7).

3.4. Blood immunity

3.5. Relative gene expression

Lysozyme showed increased activity in fish infected with A. hydrophila and fed fenugreek (Group3) compared with those in the other groups on 6 h post challenge (P < 0.05) (Table 7). After 24 h, fish in Group2 showed lower lysozyme activity compared with those in the other groups, while after 1-week post challenge fish fed fenugreek in groups 3 and 4 showed higher lysozyme activity than groups 1 and 2

Liver and kidney HSP70 showed upregulated expression in Group2 at 6 h, 24 h and 1-week post challenge (P < 0.05) (Fig. 1A and B). Liver IL-1β was significantly upregulated in fish of the 3rd group at 6 h, 24 h and 1-week post challenge (P < 0.05) (Fig. 2A), while kidney IL-1β was significantly upregulated in fish of the 2nd and 3rd group at 6 h and 1-week post challenge(P < 0.05) (Fig. 2B). 5

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(Fig. 6A, F). At 6 h post challenge there was no detectable lesions in hepatopancreas but at 24 h there was a marked change in the liver represented by small areas of hepatocytes necrosis, vacuolation, dilated and congested blood sinusoids. The lesions were exaggerated at 1-week post challenge as the necrosis became diffuse, with congested blood sinusoids and central veins (Fig. 6B and C). The hepatopancreas of fish groups fed on fenugreek showed a non-significant improvement at 24 h post challenge while at 1-week there was a mild dilatation of sinusoid, small areas of necrosis and small aggregations of melanomacrophage (Fig. 6D and E). The spleen of control and fenugreek treated groups were composed of intermingled red and white pulp in addition to the ellipsoids and melanomacrophage (Fig. 7A, F). At 6- and 24-h post challenge there was a marked necrosis and depletion of lymphocytes, presence of hemosiderin pigment and increase in melanomacrophage aggregations. The lesions were more extensive at 24 h and 1-week post challenge (Fig. 7B and C). The spleen of fish groups fed on fenugreek showed a non-significant improvement at 6- and 24-h post challenge while at 1week there was a mild depletion of lymphocytes, decrease in melanomacrophage aggregates (Fig. 7D and E). The histopathological examination of posterior part of kidney (mesonephros) of control and fenugreek treated groups revealed presence of intact glomeruli and renal tubules with melanomacrphages presence in the renal tissue (Fig. 8A, F). At 6 h post challenge the kidney didn't show any detectable lesions while at 24 h post challenge there was a marked interstitial nephritis, infiltration of inflammatory cells, necrosis of tubular cells, hyaline droplet accumulation inside renal tubules and separation of tubular cells from basement membrane. The same lesions but more exaggerated in addition to glomerulopathy and dilatation of Bowman's space was at 1-week post challenge (Fig. 8B and C). The kidney of fish groups fed on fenugreek at 24 h post challenge showed a definite infiltration of inflammatory cells, necrosis of tubular cells, hyaline droplet accumulation inside renal tubules but the lesions were less extensive at 1-week post challenge (Fig. 8D and E).

Fig. 3. Relative expression of liver and kidney TNF-α of Nile tilapia after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek for 8 weeks. Values are expressed as mean ± SE from triplicate groups. Bars with capital superscript letters indicate significant differences between groups, while small letters refer to differences within the same group and among the different sampling periods for each parameter (P < 0.05).

3.7. Disease resistance The survival rate of Nile tilapia fed with fenugreek supplemented diets for 15 days post exposure to A. hydrophila challenge are presented in Fig. 4. The data revealed that fenugreek fed groups had noticeably higher survival rate compared to the non-supplemented groups (P < 0.05). The mortality of the second group started following the 1st day post exposure to A. hydrophila while in the 1st group started after 3 days. Mortality of fish in the 3rd and 4th groups started from the 5th and 6th day, respectively. Monitoring the data exhibited that fish fed

Liver TNF-α was upregulated in the 3rd group at 6 h post challenge (P < 0.05), while the 3rd and 4th groups showed the highest levels after 24 h (P < 0.05) (Fig. 3A). After 1-week the 4th group showed the highest level of liver TNF-α. Kidney TNF-α was upregulated in the 4th group at 6 h post challenge (P < 0.05), while the 2nd and 3rd groups showed the highest levels after 24 h (P < 0.05) (Fig. 3B). After 1-week the 2nd group showed the highest level of kidney TNF-α. 3.6. Histopathological findings The anterior part of small intestine of control and fenugreek treated groups exhibited normal structure; intact tunica mucosa, submucosa, muscularis and serosa. The increase in length, branching and density of intestinal villi were evident at 1-week of feeding fenugreek (Fig. 5A, G, H). At 6 h post challenge with A. hydrophila the intestinal villi showed marked necrosis and sloughing of the apical part (Fig. 5B). The necrosis was extensive to include the whole intestinal mucosa and the exposure of lamina propria in addition to accumulation of enterocytes and tissue debris inside the intestinal lumen at 24 h and 1-week post challenge (Fig. 5C). There was a non-detectable decrease in degree of necrosis in the challenged group fed on fenugreek at 6 h while the severity of lesions was decreased at 24 h and 1-week post feeding with fenugreek (Fig. 5D, E, F). Normal hepatic architecture; the polyhedral hepatocytes radiating from central veins and separated by blood sinusoids in addition to pancreatic venous tract which composed of portal afferent vein coated by exocrine pancreatic acini were seen in control and fenugreek groups

Fig. 4. Cumulative survival of tilapia challenged with A. hydrophila after feeding basal diet only (control) and basal diet containing fenugreek for 8 weeks. The survival rate is calculated according to the Kaplan-Meier method. 6

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Fig. 5. Photomicrograph of Nile tilapia intestine after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek for 8 weeks. (A) Control group showing intact layers of intestine; tunica mucosa (E), tunica submucosa (SM), tunica muscularis (M) and tunica serosa (S). (B) Infected intestine at 6 h post challenge showing sloughing of apical part of intestinal villi (arrow head). (C) Infected intestine at 24 h post challenge showing severe necrosis and sloughing of lamina mucosa along the whole length of villi (arrow head) and exposure of lamina propria (arrow). (D) Moderate necrosis in the apical and middle part of intestinal villi (arrow head) and exposure of lamina propria (arrow) in inftected + fenugreek at 6 h post challenge. (E) Inftected + fenugreek at 24 h post challenge showing necrosis in the apical part of intestinal villi (arrow head). (F) Inftected + fenugreek at 1-week post challenge showing mild degeneration and sloughing in the apical part of intestinal villi (arrow head). (G) The intestine of fish fed on fenugreek containing ration at 24 h showing increase the density of intestinal villi. (H) The intestine of fish fed on fenugreek containing ration at 1-week showing increase in the length and branching intestinal villi (arrow), H&E, bar = 100 μm.

fenugreek had the highest relative survival rate and lowest mortality when compared with other groups.

The used medicinal plant (fenugreek) in the present study was efficient in the treatment of various human and animal diseases, being rich in flavonoids and polyphenols in addition to tannins, which contributes in enhancing the immune system and antioxidative response (Guardiola et al., 2017a, 2017b; Olaiya and Soetan, 2014). After 8 weeks, the growth performance was greatly improved by fenugreek feeding and this was synchronous with the enhancement in the main immune activities. Medicinal herbs and its extracts are considered as potential immune enhancers and antioxidants when applied in the land-based captive, hatchery and farmed animals (Van Doan et al., 2019b, 2019c). These additives could augment the growth of beneficial microbial colonies in the digestive tract which lead to enhance feed intake and weight gain of fish (Safari et al., 2017). In agreement with this study, gilthead sea bream recorded significant increase in growth rate as well as immune parameters after fed diets

4. Discussion Emergence of antibiotic-resistant strains of bacteria led to use of eco-friendly alternatives such as herbal products as a food supplement in aquaculture production (Dawood et al., 2018). Unlike synthetic chemicals, herbal supplements are degradable in addition to being inexpensive; and they do not cause resistance in pathogenic agents due to having very diverse active ingredients. Although the plenty of research for controlling Motile Aeromonas Septicemia disease in tilapia using natural products (Ardó et al., 2008; El-Asely et al., 2014), the mortalities associated with the disease outbreaks still constituting a continued important problem in tilapia farming (Fernandes et al., 2019). 7

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Fig. 6. Photomicrograph of Nile tilapia hepatopancreas after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek for 8 weeks. (A) Control group showing polygonalshaped hepatocytes (H), blood sinusods (S) and pancreatic acini(P). (B) Infected liver at 24 h post challenge showing diffuse vacuolation (arrow head), necrosis of hepatocytes (thick arrow) and mild dilatation of blood sinusoids (thin arrow). (C) Infected liver at 1-week post challenge showing severe necrosis of hepatocytes (arrow) and pancreatic cells (arrow head). (D) Marked necrosis of hepatocytes (thick arrow), congestion and dilatation of blood sinusoids (thin arrow) and central vein (CV) in inftected + fenugreek at 24 h post challenge. (E) Inftected + fenugreek at 1-week post challenge showing focal areas necrosis (thick arrow) and mild dilatation of blood sinusoids (thin arrow) in addition to small aggregates of melanomacrophage (arrow head). (F) The liver of fish fed on fenugreek containing ration at 1-week showing normal hepatic architecture, cords of hepatocytes (H) separated by blood sinusoids (S), H&E, bar = 50 μm.

supplemented with fenugreek (Awad et al., 2015). This could be attributed to the known efficiency of fenugreek seeds to improve protein digestion and fat absorption capacity (Mansour and El-Adawy, 1994). However, this affirmation has been corroborated in humans and would be well advised to be undertaken in fish studies that could prove the reason why fenugreek has such effects on fish growth. Blood biochemical and physiological markers, for example, serum compounds, could be utilized to recognize probable environmental hazards on the health of aquatic animals due to infectious diseases (Burgos-Aceves et al., 2019; Dawood et al., 2017a, 2017b; Van Doan et al., 2017). The outcomes for the enzymatic tests and metabolic items for which used in the current study exhibited significant upregulations for ALT and AST in fish challenged with A. hydrophila. The biochemical changes incited by A. hydrophila infection are because of aggravated digestion showed as restraint of catalysts, harm and brokenness of the tissues, especially liver cells (Adel et al., 2017). ALT and AST have to a great extent been accounted for the examinations of fish wellbeing, liver enrichment with proteins, irresistible sicknesses, and contamination with toxics (Racicot et al., 1975). The results obtained in this study showed high ALT and AST in the fish specimens challenged with A. hydrophila, while the lowest mean values were found in the fish fed fenugreek. Antioxidant defense response following oxidative stress resulting from pathogen entry and respiratory burst activity is important reaction, because this stress reduces meat quality through lipid peroxidation and consequently influences fish health (Aliko et al., 2018; Gobi et al., 2018). The deleterious effect of excessive ROS is balanced by many

cellular defense activities, including antioxidant defense mechanisms (Apel and Hirt, 2004; Burgos-Aceves et al., 2018). Evaluation of SOD, CAT and GPx activity as the important antioxidant enzymes can be considered as the biomarkers of oxidative stress besides indicating antioxidant capacity of aquatic organisms (Gaetani et al., 1996). The over ROS creation can increase the lipid peroxidation which resulted in malondialdehyde (MDA) production. The continuous existing of MDA can damage the organism cell by breaking down the DNA, protein and cytoplasm (Yao et al., 2010). Our results revealed lower MDA and higher SOD and GPx activities in fish fed with fenugreek indicated reduced cell damage compare to fish fed control diet. Earlier studies also showed an enhanced SOD and GPx levels by fenugreek feeding (Awad et al., 2015). The authors attributed that to the presence of bioactive compounds, which might improve the antioxidant activity and regulate the ROS intermediate system. Fenugreek has functional compounds that enhanced the secretion of antioxidant enzymes and helped in scavenging superoxide radicals. These compounds have high reducing and antioxidant properties due to the presence of hydroxyl group and unsaturated bonds in their chemical structure. Accordingly, phenolic acid and gallic acid are the potent phenolic antioxidants, which can increase the antioxidant properties of the extracts by creating hybrids with other phenolic compounds such as caffeic acid (Badhani et al., 2015). Moreover, the existence of quercetin, a flavonoid antioxidant in fenugreek due to the lack of glycogen in its structure has high antioxidant capacity (Badhani et al., 2015). The increase of antioxidant enzyme activity in the present study was in consistent with the increase of immune parameters. 8

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Fig. 7. Histopathological changes in Nile tilapia spleen after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek for 8 weeks. (A) Control group showing normal organization of spleen with ellipsoids (E), red and white pulp (P) in addition to melanomacrophage center (MMC). (B) Infected spleen at 6 h post challenge showing necrosis and depletion of lymphocytes (thick arrow) beside presence of hemosiderosis (arrow head) and increase in melanomacrophage aggregations (MMC). (C) Infected spleen at 24 h post challenge showing diffuse areas of necrosis and depletion of lymphocytes (thick arrow) beside presence of large area of hemosiderosis (arrow head). (D) Areas of necrosis (thick arrow), and accumulation of melanomacrphage (arrow head) in the spleen of inftected + fenugreek at 6 h post challenge. (E) Inftected + fenugreek at 1-week post challenge showing focal areas necrosis and depletion of lymphocytes (arrow). (F) The spleen of fish fed on fenugreek containing ration at 1-week showing normal architecture with ellipsoids (E), red and white pulp (P) in addition to melanomacrophage center (MMC), H&E, bar = 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

The non-specific immune response as the first line of defense system is especially important to protect fish against invading pathogens (Van Doan et al., 2017). Accordingly, previous researches have well approved the impacts of herbal products on improving non-specific immune response in various fish and shellfish species (Awad and Awaad, 2017). In line with previous findings, the results of our study revealed that long-term administration of fenugreek could improve humoral immunity (lysozyme activity, IgM, and NBT). In the present study, the serum lysozyme activity significantly increased in fenugreek fed groups compared to the control. Based on the study of Engstad et al. (1992), the increase in lysozyme in the blood of stimulated fish is associated either with the proliferating phagocytes or the increased the amounts of lysozymes produced from the lysosomes. Rendering lysozyme activity to be one of the best markers for evaluating the bactericidal effect of the feed additives. The increase in the lysozyme activity in our work could be due to the flavonoids which stimulates leucocytes and phagocytosis (Awad et al., 2015). Fish neutrophils contain various phagocytic, bactericidal and respiratory burst (NBT) activities (Vallejos-Vidal et al., 2016). Evaluation of the neutrophil function is necessary in the assessment of the general health of fish (Cerezuela et al., 2012). We determined, herein, that the administration of fenugreek appreciably increased the blood NBT. Similarly, gilthead seabream fed fenugreek showed enhanced NBT (Guardiola et al., 2018a, 2018b). Respiratory burst, through stimulation by foreign agents, have been found to increase the oxidation levels in phagocytes, and are a crucial factor in the general defense mechanisms

in fish (Lee et al., 2000; Miyazaki, 1998). The creation of respiratory burst activities and reactive oxygen metabolites by phagocytes are vital factor in limiting the spread of diseases in fish (Rodríguez et al., 2003). Blood total protein is associated with the influence of serum proteins (e.g. IgM) as an indicator for the enhanced immune system of fish (Magnadottir, 2010). Immunoglobulins are “heterodimeric glycoproteins that play a vital role in recognizing natural antigens and exist in the skin, gill and gut mucus, bile as well as systemically found in the plasma of fish” (Uribe et al., 2011). Enhanced blood IgM in the current study is in line with the increased lysozyme activity, suggesting immunomodulatory effects of fenugreek on Nile tilapia after A. hydrophila challenge. Similarly, gilthead seabream fed fenugreek showed enhanced IgM (Guardiola et al., 2018a, 2018b). Although, the exact mechanisms underlying the impacts of phytochemical compounds on the immune function of fish is not well understood. Nevertheless, the beneficial effects of herbal supplements on the immune function of aquatic animals have widely been attributed to the role of bioactive components in their structure. The immunomodulatory effects of fenugreek can be due to presence of a broad spectrum of active substances such as phenolic compounds (gallic acid, chlorogenic acid, vanilic acid, p-coumaric acid and ferulic acid), polyphenols as well as flavonoids (rutin, quercetin, myerstin, naringin and catechin) (Olaiya and Soetan, 2014). Accordingly, many studies have already highlighted the effects of secondary metabolites compounds on improving immune function of fish (Awad et al., 2015; Bahi et al., 2017; Guardiola et al., 2018a, 2018b; Roohi et al., 2017). The impacts of these ingredients as 9

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Fig. 8. Histopathological changes in Nile tilapia kidney (mesonephros) after 6 h, 24 h and 1-week post infection in different groups fed diets with or without fenugreek for 8 weeks. (A) Control group showing normal organization of kidney with glomeruli (G) and renal tubules (T). (B) Infected spleen at 24 h post challenge showing infiltration of inflammatory cells (black arrow head), necrosis in renal tubules (thick arrow) beside accumulation of hyaline droplets inside tubular epithelium (white arrow head) and separation of tubular epithelium from basement membrane (thin arrow). (C) Infected kidney at 1-week post challenge showing diffuse necrosis of tubular epithelium (thick arrow) and glomerulopathy (thin arrow) beside accumulation of hyaline droplets in renal tubules (white arrow head), small aggregates of melanomacrphage (white arrow). (D) Diffuse areas of necrosis (thick arrow), and infiltration of inflammatory cells (arrow head) in the kidney of inftected + fenugreek at 24 h post challenge. (E) The kidney inftected + fenugreek at 1-week post challenge showing infiltration of inflammatory cells (arrow head) small aggregates of melanomacrphage (white arrow). (F) The kidney of fish fed on fenugreek containing ration at 1-week showing normal architecture with glomruli (G) and renal tubules (T) beside small aggregates of melanomacrphage (white arrow), H&E, bar = 50 μm.

the secondary metabolites in plants are species specific, and the effectiveness of most medicinal plants depends on the diversity and quantity of these ingredients (Awad and Awaad, 2017). HSP70 is a significant tool to promote the cell survival rate through stress protection, cure and environmental pressure relief (Lindquist and Craig, 1988; Ming et al., 2010), and HSP70 relative expression is a potential stress marker for aquatic animals (Faggio et al., 2016; Forsyth et al., 1997). This research founded that after A. hydrophila challenge was initiated, fenugreek groups showed a considerably greater concentrations of expression of kidney and hepatic HSP70. This indicates that diets with fenugreek can balance HSP70 expression in fish after A. hydrophila challenge. Administering medicinal herbs alters the transcription of pro- and anti-inflammatory cytokine genes in different organs (Jiang et al., 2016). Moderate transcription of pro inflammatory cytokines is beneficial for the maintenance of immunological balance and to increase resistance against infection. Confusion may arise as to whether higher transcription of pro-inflammatory cytokines after additive supplementation leads to inflammation or immunomodulation. The roles of medicinal herbs are more or less similar to that of immunostimulants and their supplementation mainly upregulates pro-inflammatory cytokines (Safari et al., 2017). TNF-α and IL-1β are pro-inflammatory cytokines that demonstrate higher transcription levels after fenugreek supplementation in fish (Biswas et al., 2013; Xia et al., 2018). In this study, the obtained results showed upregulated immune related genes (TNF-α and IL-1β) expressions in Nile tilapia post infection with A. hydrophila, TNF-α is cytokine that mostly secreted by activated

macrophages, orchestrates immune defence mechanisms against pathogen invasion and colonisation, and stimulates neutrophil base immunity (Bilen et al., 2019). Similarly, an increased immune related gene expression was observed following fenugreek feeding in gilthead seabream (Bahi et al., 2017). IL-1β is a pro-inflammatory cytokine activating the lymphocytes and macrophages against disease-causing aetiologies (Low et al., 2003). In this study, IL-1β expression increased in fish fed on fenugreek compared to control. TNF-α also stimulate resting macrophages to secrete other cytokines, including IL-6 and IL-1β (Lauriano et al., 2016; Striz et al., 2014). Activated phagocytic cells search and engulf foreign invaders faster than when under unstimulated conditions. Induction of these genes by fenugreek supplements is similar to vaccination, as both stimulate the immune system. The main difference between these natural additives and vaccines is that the former kill or eradicate nonspecific pathogens, whereas the latter kill or eradicate specific ones. Interestingly, our results showed an increased expression of pro-inflammatory cytokines in the liver and kidney after infection with A. hydrophila, which might be related to the immunomodulatory role of fenugreek in Nile tilapia diets. Fenugreek fed fish exhibited tolerance to A. hydrophila challenge which resulted in significant reduced mortalities compared to fish fed diets without fenugreek. The improved survival rates in the present study can be attributed to the beneficial impacts of fenugreek on immune function as well as antioxidant capacity. It was claimed by Jiang et al. (2016) that A. hydrophila pathogen is highly correlated with the oxidative stress, from that concept the protective effect of fenugreek may be associated with richness in flavonoids and polyphenol in 10

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addition to the high concentrations of ascorbic acids which neutralize the excessive free radicals (Abdallah et al., 2016). Histopathology is widely used to study the pathological alterations due to various chemicals or biological infectious agent as biological markers (Camargo and Martinez, 2007; Forouhar Vajargah et al., 2018). In addition, head kidney, spleen and liver are involved in fish immunity through the inclusion of immune cells within its tissues (Zapata et al., 2006). In the current study, addition of fenugreek to the fish's diet reduced the pathological changes associated with A. hydrophila infection in all examined organs with an obvious effect in Group3. At the same context, Ostaszewska et al. (2008) have shown an improvement in the hepatocytes structure of the silver bream (Vimba vimba) fed on natural feed additives. The same to our findings, Owatari et al. (2018) proved that sylimarin significantly reduced the pathological alterations of the liver and spleen of Nile tilapia infected with Streptococcus agalactiae. The damaging effect of the bacterial infection in the host tissues is in part associated with the oxidative stress induction (Tkachenko et al., 2014). The mode of action of most flavonoids containing plants becomes the magic pullet; where its ability in reducing the ROS and consequently relieving the harmful effect (Brunetti et al., 2013). MMCʼs are pigmented macrophages aggregations in the kidney and spleen associated with immunity, through phagocytosis to foreign materials (Steinel and Bolnick, 2017). Nearly like our results, an increase in the number of MMC was recorded in Nile tilapia fed Echinacea purpurea supplemented diet (El-Asely et al., 2012) and in polyphenol-enriched feed administered farmed sea bass (Magrone et al., 2016).

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