Introduction

Bacterial diseases in cultured fish are considered the main problem to aquaculture system in Egypt as they induced mortalities and economic losses (Noor El-Deen et al. 2010). Motile Aeromonas Septicemia (MAS) is a serious problem for fish farming industry in Egypt as well as in other countries (Noor El-Deen et al. 2010 and Noga 2010). It affected various species of freshwater, brackish, and marine fish causing high economic losses in fish farming industry (Noga 2010). Also, the causative agent of MAS, Aeromonas hydrophila (A. hydrophila) is a zoonotic pathogen for humans causing illness that ranged from mild to severe dysentery-like diarrhea, meningitis, and septicemia (Inglis et al. 1993). It caused severe histopathological lesions in many organs including gills, intestine, and liver (AlYahya et al. 2018) in the form of inflammation, degeneration, and proliferation (Stratev et al. 2015).

Conventional approaches for prevention or treatment of bacterial fish diseases are based on the use of antimicrobial drugs that have potential hazards to public health and environment due to emergence of drug-resistant microorganisms and antibiotic residues (Sugita et al. 1990). Attention has been focused on detecting novel products, especially of plant origin for development of safe, economic, and environmental friendly alternative substances in fish disease management. Among these plants is Moringa oleifera (M. oleifera) which is a highly valued plant and distributed in tropical and subtropical countries. It has a wide range of medicinal uses with high nutritional value, the so called “miracle tree” (Ramachandran et al. 2014). Antimicrobial activities of various M. oleifera morphological parts against some pathogenic microorganisms have been reported (Vieira et al. 2010). Moringa oleifera leaves (MOLs) extract was used successfully in treatment of MAS disease among infected Clarias gariepinus (Hammed et al., 2015). Only few studies had been carried out to evaluate the effectiveness of M. oleifera on prevention of MAS disease in fish. Thus, the present study was focused on evaluation of MOLs and their aqueous extract as feed additives in prevention of MAS among C. carpio fingerlings. Also, detection of histopathological alternations of fish groups fed on MOLs and their aqueous extract in comparison with the control positive and negative groups was performed.

Material and methods

Fish collection and maintenance

A total of 150 apparently healthy C. carpio fingerlings with average body weight 7 ± 2.0 g were collected for determination of median lethal concentration (LD50) of MAS. Also, a total of 180 apparently healthy C. carpio fingerlings with average body weight 7 ± 2.0 g were used in the prevention of MAS experiments. On the other hand, a total of 180 apparently healthy C. carpio fingerlings with average body weight 7 ± 2.0 g were needed for histopathological examination. All experimental fish were collected from Beni-Suef Abo-Saleh fish hatchery, Egypt, and transferred in plastic bags containing oxygenated water to the wet laboratory of Fish Diseases and Management Department, Faculty of Veterinary Medicine, Beni-Suef University, Egypt. Fish were kept in fiberglass tanks of 400 l capacity for each, supplied with chlorine-free tap water and continuous aeration. The fingerlings were acclimatized for 14 days in the experimental fiberglass tanks and were fed 5% body weight pelleted commercial fish diet (Brsiek factory, Egypt, Table 1) during acclimation.

Table 1 Composition of the commercial pelleted fish diet (Brsiek factory, Egypt)
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After acclimatization, fish were redistributed into glass aquaria of 70 × 25 × 40 cm (ten fish/each) for running the experiments. Parameters of water quality were measured twice a week during acclimatization and throughout the experimental periods. These parameters include measuring dissolved oxygen using DO meter (Yellow Spring Instrument Co., Yellow Springs, Ohio, USA), temperature using water thermometer (Yellow Spring Instrument Co., Yellow Springs, Ohio, USA), and pH by PH indicator paper (Fisher Scientific, Denver, Colorado, USA), as well as measuring ammonia, nitrite, and nitrate using commercial test kits (Aquamerck; Merck, Darmstadt, Germany).

Source of MOLs

Moringa oleifera leaves were obtained from local market. The specimen was examined and identified by a botanist from Botany Department, Faculty of Sciences, Beni-Suef University, Egypt. The obtained leaves were washed with distilled water and air dried in shaded area. The dried leaves were grinded into fine powder using mixer and stored in sterilized glass containers at room temperature for use.

Preparation of aqueous MOLs extract

The aqueous extract of MOLs was obtained according to the method described by Fatope et al. (1993) and Khalil and Korni et al. (2017). Twenty-five grams of powder of grinded leaves was mixed with 250 ml hot (98 °C) distilled water and kept for 24 h. The extract was filtered using a muslin cloth and then re-filtered using filter paper. The extracts were labeled and preserved in a refrigerator at 4 °C then used within 1 week.

Diet preparation

The pelleted commercial fish diet (Brsiek factory, Egypt, Table 1) was ground into fine powder by using mortar. The MOLs and its aqueous form were mixed separately with the fine powder. Three fish diets were prepared, including diet1 (d1) with no additives (control), diet2 (d2) containing 1 g of MOLs/kg feed, and the third diet (d3) containing 10 ml of 10% MOLs aqueous extract/kg feed. Each fish diet contents were mixed with distilled water for obtaining a homogenous mixture. The mixture was passed through a hand minced-meat processing machine to produce extruded strings, which were dried at room temperature for 24 h and then broken down to small pellets.

Experimental design

Estimation of the antimicrobial activity of MOLs aqueous extract for prevention of MAS

MIC of MOLs aqueous extract

The lowest concentration Minimum Inhibitory Concentration (MIC) of MOLs aqueous extract that inhibits the growth of A. hydrophila pathogenic strain was estimated by the broth microdilution method according to Wikler (2000). Freshly prepared broth culture of pathogenic A. hydrophila strain was maintained at an optical density of 0.1 at 600 nm (OD of 0.1 corresponds to a concentration of 108 CFU ml−1). Each 1 ml of broth culture was supplemented with 10,000, 5000, 2500, 1250, 625, and 312 micro/ml of prepared aqueous solution in Mueller-Hinton broth. Culture broth used without aqueous solution but with the bacterial culture served as control negative, while another one which used with last concentration of aqueous solution and without bacterial culture was served as control positive. All treatments and controls were incubated at 37 °C for 24 h. The experiments were carried out in triplicate. Evaluation of the MIC was carried out by the last tube which showed visually no turbidity or bacterial growth. Those are confirmed by the MBC test and the colony number was converted to the concentration of bacterial cells (CFU ml−1) as the method described by CLSI (2006) and Perez et al. (1990).

MBC of MOLs aqueous extract

The minimum bactericidal concentrations (MBCs) were determined by inoculating 10 μL from the broth culture with no visible growth, the previous tube, and followed one onto Mueller-Hinton agar then incubated at 37 °C for 24 h. The MBC was the plate of aqueous concentration that completely causes bactericidal growth and no colonies on the agar plates.

Disc diffusion assay of the MOLs aqueous extract

Disc diffusion assay was performed by using Whitman filter paper with standard size of 50 mm diameter obtained by punching and kept in capped sterile containers. The containers were sterilized in a hot air oven at 150 °C for 30 min. The standard sterilized discs were impregnated overnight with the two different tested concentrations of MOLs aqueous solution (1000 micro/ml). Bacterial isolates were diluted in normal saline at a concentration of 108 CFU mL−1 according to reference to McFarland 0.5 (1.5 × 108). Thereafter, 100 μL of a suspension was streaked on Mueller–Hinton agar plates. The impregnated discs were placed aseptically with some sterile forceps on Mueller–Hinton agar plates in the presence of the same discs in the standard antibiotic difloxacin 92.9% (Pharma Swede Pharmaceutical Company). All plates were incubated at 37 °C for 24 h. The inhibition zone diameter of all tested bacteria was determined via estimation of the two diameters mean of the zone (length and width) by the help of graduated ruler. All the readings were taken in triplicate (CLSI, 2006 and Perez et al. 1990).

Prevention of MAS in C. carpio fingerlings

Bacterial strain

The causative agent of MAS (pathogenic strain of Aeromonas hydrophila) was obtained from previous study of diseased O. niloticus and identified by PCR for detection of hemolysin virulence gene (Korni et al. 2017).

Determination of LD50 of A. hydrophila isolate in healthy C. carpio

A total of 150 apparently healthy C. carpio fingerlings were subdivided into five groups (10 fish/each) with three replicates. An overnight culture of the isolate was adjusted to densities of 1.5 × 10,7 1.5 × 10,6 1.5 × 105, and 1.5 × 104. Each dilution was injected at a dose of 100 μl/fish intraperitoneally, and the fish of the fifth group were injected with 100 μl of physiological saline (control group). All fish groups were observed for 2 weeks. Mortalities were recorded daily, and the organs were aseptically streaked on TSA agar for re-isolation and re-identification.

Prevention scheme

After acclimatization of 180 fingerlings, they were divided into six groups (10 fish/each) with three replicates. Fish in the first and second groups were fed on d1, while fish in the third and fourth groups were fed on d2. Additionally, fish in the fifth and sixth groups were fed on d3. Throughout the experimental period, the fingerlings were fed 5% of body weight with its specific diet once a day at 10 a.m. for 30 days.

At the end of dietary experimental period (30 days), the first (control positive), third, and fifth groups with their replicates were challenged intraperitoneally with pathogenic A. hydrophila strain at a dose of 100 μl of 1.5 × 106 concentration. On the other hand, the second (control negative), fourth, and sixth groups were injected intraperitoneally with 100 μl physiological saline. The injected fingerlings were maintained in a separate glass aquaria for 2 weeks. The mortalities were recorded and the relative percent survival (RPS) was calculated according to Amend (1981) using the following formula:

$$ \mathrm{RPS}=1-\left(\%\mathrm{of}\ \mathrm{mortality}\ \mathrm{in}\ \mathrm{treated}\ \mathrm{group}\mathrm{s}/\%\mathrm{of}\ \mathrm{mortality}\ \mathrm{in}\ \mathrm{control}\ \mathrm{group}\right)\times 100. $$

Histopathological examination

After acclimatization of 180 fingerlings, they were divided into six groups (10 fish/each) with three replicates. Fish groups were fed and injected as the same manner of the previous prevention scheme. Ninety-six hours post injection, the fish of each feeding group were anesthetized by MS 222; the gills, skeletal musculature, liver, intestine, and kidney of each group were collected separately and fixed in 10% neutral buffered formalin at room temperature. Samples were trimmed for a size of one cubic centimeter. The collected specimens underwent routine histological procedures: dehydration using ascending grades of alcohol, clearing using xylene, and paraplast embedding at 56 °C in hot air oven. Sections of 4–6 μm thickness were obtained then stained with hematoxylin and eosin and examined by the light microscopy (Bancroft and Gamble 2008).

Statistical analyses

Statistical analyses were done using all data one-way ANOVA (post hoc test; Dunnett’s test) Advanced Models 16.0 software (SPSS, Tokyo, Japan). P < 0.05 was considered as statistically significant.

Results

Antibacterial assay

The measured zone of inhibition showed that MOLs aqueous extract has an important bactericidal activity against pathogenic A. hydrophila. The diameters of the zones increased with increasing the aqueous concentrations at bacterial dilutions (Table 2; Fig. 1). Results showed that MOLs aqueous extract exhibited inhibition zone (mm) of about 35.5 till 29.25 mm in diameter for 10,000 μg/ml and 2000 μg/ml compared with standard difloxacin antibiotic (ranged from 39.5 till 33.25 mm).

Table 2 The zone of inhibition in mm for the MOLs aqueous extract against pathogenic A. hydrophila strain
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Fig. 1
figure 1

Zone of inhibition of MOLs aqueous extract against pathogenic A. hydrophila strain in the presence of standard difloxacin antibiotic

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MIC and MBC assay results were recorded in terms of MIC, which is the lowest concentration of antimicrobial agent causing almost complete inhibition of growth or giving no visible growth. MIC and MBC values ranged from 12 to 24 μg/ml respectively.

Median lethal dose (LD50)

The mortality of the experimentally infected C. carpio fingerlings was reported for 2 weeks after intraperitoneal injection with different concentrations of pathogenic A. hydrophila isolate. The fish death occurred during the first week of the experiment, and the LD50 was 1.5 × 105 CFU/ml.

Prevention of MAS in C. carpio fingerlings

The group of fish fed MOLs and their aqueous extract appeared and behaved similar to those reared with the control diet without any supplement. Furthermore, no mortalities and 100% RPS were observed in the group fed MOLs aqueous extract then injected with A. hydrophila, group fed MOLs aqueous extract then injected with physiological saline, group fed MOLs then injected with physiological saline, and group fed normal diet then injected with physiological saline (control negative). Contrarily, there were 10% mortalities and 86 RPS in group fed MOLs then injected with A. hydrophila. On the other hand, the control positive group showed 70% mortalities.

Histopathological alterations

Group of C. carpio fingerlings fed normal diet for 30days (control negative) then injected with saline

The gills showed normal histological structure of gill filaments, lamellae, and arch (Fig. 2a).The skeletal muscles showed normal histological structure of the muscle bundles with prominent cross striation (Fig. 2b). The liver revealed normal histological picture in the form of normal arrangement of hepatocytes, blood sinusoids, central vein and portal area with the pancreatic structure (Fig. 2c).The intestine showed no histopathological alternations with normal histological structure of the intestinal layers, mucosa, submucosa, musclosa, and serosa (Fig. 2d). The kidney showed normal histological structure of the renal corpuscles and renal tubules (Fig. 2e).

Fig. 2
figure 2

A photomicrograph of the control negative group of C. carpio fingerlings. a Gills showing normal histological structure of the gill filaments, gill lamellae, blood capillaries, and lining epithelium, b Skeletal muscles showing ordinary muscle bundles with prominent cross striation. c Liver showing normal arrangement and structure of the hepatocytes, blood sinusoids, central vein, and portal area as well as pancreatic structure. d Intestine showing normal histological structure of the intestinal lining epithelium (columnar cells and goblet cells), submucosa, musclosa, and serosa. e Kidney showing normal histological picture of the renal glomeruli and tubules. H&E stain; × 100, × 400, × 100, × 100, and × 100, respectively

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Group of C. carpio fingerlings fed normal diet for 30 days then injected with A. hydrophila strain (control positive)

The gills showed degenerative changes with sloughing of the lining epithelium, congestion of the blood vessels, and massive infiltration of inflammatory cells in the gill lamellae (Fig. 3a).The skeletal muscles revealed degenerative changes and massive infiltration of inflammatory cells as well as alteration of the cross striation (Fig. 3b). Severe degenerative changes in the hepatocytes, congestion of the central vein, and inflammatory cell infiltration in the blood sinusoids were observed in liver. In addition, the portal area showed edema and many inflammatory cells in between the pancreatic structure (Fig. 3c). The intestine showed degenerative changes and sloughing of the lining epithelium, congestion of the blood vessels in the submucosa accompanied with massive inflammatory cell infiltration (Fig. 3d). The kidney revealed degenerative changes in the renal tubules and inflammatory cells infiltration in between the renal tubules (Fig. 3d).

Fig. 3
figure 3

A photomicrograph of control positive group of C. carpio fingerlings. a Gills with degenerative changes and sloughing of the lining epithelium, congestion of the blood vessels, and massive infiltration of inflammatory cells in the gill lamellae. b Degenerative changes in the muscle bundles with infiltration of inflammatory cells. Note: alteration of the cross striation in the muscle cells. c Liver showing degeneration of hepatocytes, congestion of the central vein, and inflammatory cells in the blood sinusoids. Note: portal area showed edema with inflammatory cell infiltration in between the pancreatic structure. d Degenerative changes and sloughing of the intestinal lining epithelium, congestion of the blood vessels in the submucosa accompanied with massive infiltration of inflammatory cells. e Degenerative changes in the renal tubules and accumulation of inflammatory cells in between the renal tubules. H&E stain; × 200, × 400, × 400, × 200, and × 200, respectively

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Group of C. carpio fingerlings fed MOLs for 30 days then injected with saline

The examined organs of this group showed normal histological pictures (Fig. 4a–e).

Fig. 4
figure 4

A photomicrograph of group of C. carpio fingerlings fed MOLs for 30 days. a Normal histological structure of gills. b Normal histological picture of muscles. c Normal histological architecture of liver. d Normal picture of intestinal layers. e Normal histological structure of kidney. H&E stain; × 200, × 400, × 200, × 200, and × 400, respectively

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Group of C. carpio fingerlings fed MOLs for 30 days then injected with A. hydrophila strain

Gills, muscles, and intestine of this group showed normal histological architecture (Fig. 5a, b, d). However, liver showed minor hydropic degeneration of the hepatocytes and few inflammatory cells in the blood sinusoids (Fig. 5c). Also, kidney showed minor hydropic degeneration of the renal tubules with few inflammatory cells infiltrated between renal tubules (Fig. 5e).

Fig. 5
figure 5

A photomicrograph of group of C. carpio fingerlings fed MOLs for 30 days then injected with the causative agent of MAS disease, A. hydrophila strain. a Normal histological picture of gills. b Normal histological structure of muscles. c Minor hydropic degeneration of the hepatocytes and few inflammatory cell infiltration in the blood sinusoids. d Normal histological architecture of intestine. e Minor hydropic degeneration of the renal tubules with few inflammatory cells between renal tubules. H&E stain; × 200, × 400, × 400, × 200, and × 400, respectively

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Group of C. carpio fingerlings fed aqueous extract of MOLs for 30 days then injected with saline

The examined organs of this group showed normal histological architecture (Fig. 6a–e).

Fig. 6
figure 6

A photomicrograph of group of C. carpio fingerlings fed aqueous extract of MOLs for 30 days. a Normal histological structure of gills. b Normal histological picture of muscles. c Normal histological architecture of liver. d Normal picture of intestinal layers. e Normal histological structure of kidney. H&E stain; × 200, × 400, × 200, × 200, × 400 respectively

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Group of C. carpio fingerlings fed MOLs aqueous extract for 30 days then injected with A. hydrophila strain

Gills, skeletal musculature, liver, intestine, and kidney showed no histopathological alternations (Fig. 7a–e).

Fig. 7
figure 7

A photomicrograph of group of C. carpio fingerlings fed aqueous extract of MOLs for 30 days then injected with the causative agent of MAS disease, A. hydrophila strain. a Normal histological structure of gills. b Normal histological picture of muscles. c Normal histological architecture of liver. d Normal histological picture of the intestine. e Normal histological structure of kidney. H&E stain; × 200, × 400, × 400, × 200, × 400 respectively

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Discussion

Motile Aeromonas septicemia is one of the most common septicemic bacterial diseases of freshwater fish causing severe economic losses in aquaculture production. The continuous search for valuable medicinal plants is in progress scientifically in order to combat the surge of antimicrobial resistance (Ajayi and Fadeyi 2015). In the current study, the antimicrobial activity of MOLs aqueous extract was evaluated against pathogenic A. hydrophila strain in vitro. Also, dietary incorporation of MOLs and their aqueous extract was evaluated for prevention of MAS disease in C. carpio fingerlings and the histopathological examination was performed. Moringa oleifera leaves aqueous extract showed great antimicrobial activity against pathogenic A. hydrophila strain. This activity might be attributed to the presence of flavonoids, saponins, tannis, polyphenols, and alkaloids that are responsible for the antimicrobial activity of MOLs aqueous extracts (Patil and Jane 2013). Previous studies confirmed the antimicrobial activity of plants containing phenolic compounds as these compounds are responsible for the deprivation of iron or hydrogen from binding with vital proteins of the microbial enzymes formation. Also, the presence of short peptides in the plant act directly on the inhibition of the bacterial growth by disrupting cell membrane synthesis or synthesis of essential enzymes (Silvestro et al. 2000). The results of this study were supported by Vieira et al. (2010) and Kalpana et al. (2013) who confirmed the bactericidal activity of MOLs aqueous extract against both Gram-positive and Gram-negative microorganisms. The current study revealed that MOLs aqueous extract exhibited antimicrobial effect at varying degrees according to the different concentrations; this may be indicative that MOLs aqueous extract can be used efficiently for prevention of MAS in fish. The in vitro antimicrobial results were confirmed by in vivo study, whereas there were 0% and 10% mortalities in groups of fish fed MOLs aqueous extract and MOLs respectively then injected with A. hydrophila pathogenic strain in comparison with 70% mortalities in control positive group. These results were augmented by Hammed et al. (2015) who proved that MOLs extract could be used successfully for treatment of MAS in infected C. gariepinus at 50% concentration without adverse effect.

The success of MOLs and their aqueous extract in prevention of MAS disease in C. carpio fingerlings might be attributed to their immune stimulant effect and mitigation of the adverse effects of stressors in C. carpio fingerlings (Khalil and Korni et al. 2017) as A. hydrophila is an opportunistic pathogen causing disease in fish under stress (Stratev and Odeyemi 2016). The superior effect of MOLs aqueous extract may be related to its more marked effect on fish performance and stress resistance than MOLs (Khalil and Korni et al. 2017).

For histopathological findings, experimental infection with A. hydrophila caused histopathological lesions in skeletal musculature, gills, liver, intestine and kidney (control positive group) in the form of edema, degenerative changes, and inflammatory cell infiltration. These results were in harmony with Harikrishnan et al. (2009) who observed that A. hydrophila caused histopathological alternations in the gold fish. Stratev et al. (2015) noticed that the most histopathological damages caused by A. hydrophila were observed in liver and kidney followed by intestine and muscles. Moreover, AlYahya et al. (2018) showed that the typical histopathological alterations were observed in liver, gills, and intestine.

The obtained histopathological lesions may be attributed to toxins and other extracellular products (hemolysin, elastase and protease) expressed by A. hydrophila (AlYahya et al., 2018). These extracellular products resulted in severe damages of tissues inducing loss of their structural integrity (Rey et al. 2009) and shared in the development of bacterial disease in fish (Chopra et al. 2000). Mild histopathological alternations that were detected in fish group fed MOLs then injected with A. hydrophila pathogenic strain might explain the 10% mortalities induced in this group. However, the group of C. carpio fingerlings fed aqueous extracts of MOLs for 30 days then injected with A. hydrophila pathological strain had no histopathological changes in all examined organs. The obtained results were in agreement with Kaleeswaran et al. (2011) who reported presence of nitrite, glycosides, and quercetin in Morenga oleifera plant enhances the hepato-protection and immunity against microbial infection and oxidative stress. Also, Hammed et al. (2015) detected potent antioxidant in MOLs increases the antibody production against infection and keeps the normal structural integrity of different fish organs under stress conditions. Moreover, the current histopathological findings augmented by Stohs and Hartman (2015) reported different biological activities of Moringa oleifera extract including antioxidant and tissue protective. Therefore, supplementation of MOLs in fish diet under study reduced the negative effect of stressors and kept to a great extent the normal architecture of different organs; on the other hand, the supplementation of MOLs extract eliminated the side effects of stressors, enhanced the immunity, and kept the normal histological picture of different organs.

Conclusions

  1. 1-

    The current study revealed the success of MOLs and their aqueous extract in prevention of MAS in C. carpio fingerlings with the superior effect of aqueous extract. No mortalities, 100% RPS, and normal histological architecture of all examined organs were observed in group of C. carpio fed MOLs aqueous extract then injected with A. hydrophila. On the other hand, 10% mortalities and mild histopathological alternation of liver and kidney were detected in fish group fed MOLs then injected with A. hydrophila comparing with 70% mortalities and severe histopathological alternations in control positive group.

  2. 2-

    Supplementation of MOLs extract in fish diet eliminates the negative effect of stressors and kept the normal histological architecture of different organs.

Recommendations

Further studies are needed for comparing between the traditional form of MOLs and their aqueous extract and their nanoparticles form in prevention of MAS disease in fish. Also, further studies are needed to explain the superior effect of MOLs aqueous extract in MAS prevention.