The effect of chlorella vulgaris extract on Edwardsiella tarda infection in carp fish

Enas A. Khoris¹ and Soad S. Belih².

¹Fish disease unit, Animal Health Research Institute (AHRI), Tanta provincial lab., Agricultural Research Center (ARC), Egypt.

²Clinical Pathology unit, Animal Health Research Institute (AHRI), Tanta provincial lab., Agricultural Research Center (ARC), Egypt.

Abstract

The current evaluation was done for determination of the efficacy of the dietary supplementation of chlorella vulgaris ethanolic extract on the various indices of growth performance, some biochemical parameters of blood, lysozymal activity, digestive enzymes activity as well as survivability of common carp (Cyprinus carpio) fish infected with E. tarda challenge. Where about 150 carps (70±3 g), were divided at randomly into 5 experimental groups in triplicate, Gp1: negative-control. Gp2: infected control, carps fed control diet for 40 days then challenged with IP injection with (1x10⁷ CFU ml^-1) of E. tarda strain. Gp3: carps fed supplemented diet with 0.1 g/ kg chlorella extract for 40 days then challenged. Gp4: carps were fed supplemented diet with 0.2 g/ kg chlorella extract for 40 days then challenged. Gp5: carps were fed a commercial control diet and challenged at the day 40 then treated with oxytetracycline with a dose (55mg/kg of fish body weight) for 10 days. After 40 days of supplementation, an improved growth indices were observed in the supplemented groups especially Gp4. Total protein, albumin, globulin, lipase and amylase activities were significantly increased in all supplemented groups compared with the control, moreover lysozymal activity were increased in these groups throughout the experimental period. There is no a significant difference in levels of ALP, ALT, AST, creatinine, and Urea were recorded in supplemented groups and the normal levels of Gp1. The survivability of carps challenged with E. tarda was elevated from 53.3% at Gp2 up to 90, 86.6 and 80% at Gp5, Gp4 and Gp3 respectively.

So chlorella vulgaris extract is considered an optimal choice for dietry supplementation in fish farming for enhancement of growth rate, immunological status, disease resistance capability and survivability.

Keywords: Carp, chlorella vulgaris, Edwardsiella tarda, survival, infection, growth indices.

Introduction

Fish is one of the available and inexpensive sources of high-quality animal protein, fatty acid and several essential nutrients (Abbas et al. 2020)

Common carp (Cyprinus Carpio), has been farmed for over 2,000 years, it is one of the most commercially important and greatly cultivated freshwater fish in the world, contributing to 11% of the total world freshwater farming production (FAO 2010). Disease is considered one of the main obstacles to aquaculture, and it hinders economic and socio-economic development in various countries of the world (Mishra et al. 2017). Bacterial pathogens are the most economically significant etiological agents causing outbreaks in fish farming

Farmed fish are susceptible to many bacterial diseases. E. tarda is the main cause of a serious systemic disease called Edwardsiellosis, which is one of the most important bacterial diseases causing mass mortality in the various species of fish in freshwater and marine fishes of both wild and farmed population all over the world (Enany et al. 2018). Edwardsiellosis is an acute to chronic systemic disease in fish characterized by ascites, exophthalmia, hernia, and severe lesions of the visceral organs. (Rolando et al. 2022).

 For a long time, antibiotics have been widely used as a feed supplement to improve growth performance and control of microbial infectious diseases in commercial fish farming (Faggio et al. 2015). As a result of the high cost of antimicrobial chemicals, in addition to their side-effects the trend towards using safe natural alternatives has been encrouged all over the world. Using of feed additives and immuno-stimulants in aquaculture has received a considerable attention in recent years. Microalga is a safe alternative and environmentally sustainable source of feed ingredients (Pradhan et al. 2023). The two most popular microalgae are Chlorella vulgaris and Spirulina platensis, which have been reported as a potential alternative source to fishmeal because of their economic cost and environmental safety, as they contain all nutrients required for fish growth (Andrade et al. 2018).  Chlorella vulgaris is widely distributed in nature, especially in freshwater bodies. It has been proven that Chlorella vulgaris contains a high percentage of proteins, fats, polysaccharides, minerals, vitamins and other nutrients, including substances of significant bioactivity (Masojidek et al. 2011; Xu et al. 2014; Glencross et al. 2020).

1. vulgarisenhance the growth rate, feed utilization, and digestive enzymatic activities in fish when used as a feed additive (Radhakrishnan et al., 2015; Pakravan et al. 2018; Karapanagiotidis et al. 2022). Adding of C. vulgaris as a supplementto diets also increases the antioxidant capacity, innate immunity, and disease resistance of fish (Abdel-Tawwabet al. 2022). 

The purpose of this evaluation was to estimate the impact of dietary supplementation of chlorella vulgaris extract on growth indices, some biochemical parameters of blood, lysozymal activity, digestive enzymes activity as well as survivability of common carp (Cyprinus carpio) fish infected with E. tarda

Materials and Methods

Antimicrobial assays

-Antibacterial susceptibility test For E. tarda (AST).

Susceptibility testing of the present E. tarda isolate (which obtained from Poultry and Fish Diseases Department, Fac. Vet. Med. Alex. Univ. Egypt) to the following antibiotics (Amoxicillin (10µg), streptomycin (S 10 µg), Sulphamethoxazole-Trimethoprim (Cotrimoxazole) COT-(25 μg) oxytetracycline (30 µg), Gentamicin GEN- (10 μg), Norfloxacin (10µg), and enrofloxacin (5 µg). were plated on Mueller and Hinton agar plate using disk diffusion method according Clinical and Laboratory Standards Institute (CLSI) (2015).  Results were inferred as susceptible, intermediate or resistant by measuring the inhibitory zone diameter (Clinical and Laboratory Standars Institute (CLSI) 2020).

-Disc Diffusion Method

The antimicrobial properties of chlorella extract were tested against pathogenic strain of E. tarda by disc diffusion method as described by Hudzicki (2009). One mL of fresh bacterial culture (at a concentration equivalent to 0.5 McFarland) was pipetted in the centre of sterile Petri dish. Molten cooled Muller Hinton agar (MHA) was then poured into the Petri dish containing the inoculum and mixed well. Upon solidification, sterile paper disks (5 mm in diameter), that inoculated with 10 µL of chlorella suspension (Chlorella was dissolved in 5% Tween 20 and 10% Dimethylsulfoxide).  Then, the plates were incubated at 26°C for 24h. Oxytetracycline (30 µg) and Gentamicin GEN- (10 μg), were tested as positive controls (according to the result of antibacterial sensitivity test). Antimicrobial activity was detected by measuring the zone of clearance (Inhibitory zone) appeared after the incubation period. 

-Determination of minimum inhibitory concentration (MIC)

The effect of antimicrobial activity of chlorella extract was also tested using MIC. Chlorella was dissolved in 5% Tween 20 and 10% Dimethylsulfoxide (DMSO). The MIC assay was definited by using micro broth dilution test in a 96 well microtitre plate according to CLSI (2015).

The stock was two-fold serially diluted to a final dilution of 0.19 mL. bacterial suspension of E. tarda were prepared to a density to 0.5 McFarland standards. Both negative and positive control wells were performed. Dimethylsulfoxide (DMSO, Sigma) was used to indicate the presence of uninhibited bacterial growth in each well. MIC was the lowest concentration that showed no turbidity.

All assays were performed in triplicates.

Preparation of the experimental diet

Chlorella vulgaris ethanolic extract was purchased from Animalia farm, Egypt. And supplemented in the basal formulation at levels of 0.1g and 0.2 g/ kg diet (Govindharajan and Vairakannu 2021).

 The basal commercial diet used as a control diet.

Experimental design

About 180 uninfected common carps (Cyprinus carpio), which were apparently with a normal healthy condition and reflexes were taken from a local farm at Kafr El-Sheikh Governorate in Egypt. The average of body weights of fish at 0 day of the experiment was (70±3 g), carps were adapted in Laboratory condition for 15 days before the beginning of the experiment. The internal organs of 10 carps were tested bacteriologically to ensure that they were free from any systemic E. tarda infection. Then about 150 fish were divided into 5 triplicate groups (Gp) (10fish/aquarium). Gp1: negative-control, carps were fed control diet for 40 days then injected intraperitoneally with 0.1 ml of 0.9% sterile saline solution. Gp2: infected control, where carps were fed control diet for 40 days then the challenge evaluation was done by intraperitoneal injection of carps with (1x10⁷ CFU ml^-1) of E. tarda strain, at the day 40 of the experiment Gp3: carps were fed supplemented diet with 0.1 g/ kg chlorella extract for 40 days then fishes were challenged with E. tarda as before. Gp4: carps were fed supplemented diet with 0.2 g/ kg chlorella extract for 40 days then fishes were challenged. Gp5: carps were fed commercial control diet and challenged at the day 40 then treated after the first appearance of the clinical signs of infection with oxytetracycline with a dose (55mg/kg of fish bodyweight for 10 days as food additive) (Hashiem and Abd El-Galil 2012). Weight of carps of each group were taken weekly for adjusting the amount of daily consumption as 3% of the total body weight, and were fed twice daily at 8:30 and 14:30 h. The 5 groups subjected to the same management (25 ± 2 ^◦C water temperature, aerating occurred by air pumps, water exchanging with about 30% of the aquarium occurred every 3 days, with siphoning of the fecal matter). All challenged carps were inspected for about 7 days post-inoculation for the appearance of the clinical signs. Mortalities were recorded every day to determine the rate of survival, freshly dead carps were removed and subjected to the post-mortem examination.

Challenge with Edwardsiella tarda:

A virulent Edwardsiella tarda strain that identified previously was obtained from Poultry and Fish Diseases Department, Fac. Vet. Med. Alex. Univ. Egypt. And used for the challenge evaluation after 40 days of chlorella supplementation, as carps of all groups except Gp1 were intraperitoneally injected with the LD₅₀ dose of E. tarda strain (1x10⁷ CFU ml^-1.), the dose of LD₅₀ was estimated before the day of challenge by 10 days (Pandey et al. 2021). Survival and mortalities of carps were recorded for 10 days after the challenge.

Growth indices and survival rate calculations:

Weight of carps of all groups were recorded at the days 0 (initial weight), 20 and 40 (final weight), of the experiment, to estimate the growth performance indices. Carps were starved for 24h before weighing.

Indices were calculated as follow; Weight gain (g) = final fish wt (g) − initial fish wt(g) Weight gain (%) = (final fish wt − initial fish wt)/initial fish wt × 100. specific growth rate (SGR) = 100 × [(Ln final fish wt) − (Ln initial fish wt.)]/days fed. Feed conversion ratio (FCR) = feed intake (g)/wt gain (g). Fish survival % = 100 × final number/initial number.

 Blood samples

Blood samples were taken two times from all groups; at the day 40 of the feeding trial and post-challenge (at the end of the experiment, in the 55th day). According to Stoskopf (1993). Carps were starved 24 hours immediately before taking samples of blood. Fishes (9 fish/group) were anaesthetized with 50 mg/L of benzocaine solution. Blood samples were drawn from the caudal vein of fishes and the samples were taken in clean dry centrifuge tube without anticoagulant. The collected blood was centrifuged at 5000 rpm for 5 minutes at room temperature for serum collection and stored at -20°C for further assays.

Determination of serum biochemical parameters:

 Some serum biochemical investigations were measured by enzymatic methods using an automated analyzer like, hepatic and kidney health indicators: aspartate and alanine aminotransferases (AST and ALT) activities (Reitman and Frankel 1957), Alkaline phosphatase (ALP) (Tietz 1995), creatinine (Houot 1985), urea (Batton and Crouch 1977). And total protein (TP) (Doumas et al. 1981), albumin (Reinhold 1953).  Globulin was calculated mathematically by subtraction of albumin value from TP, and albumin/ globulin ratio was also estimated by dividing both values.

 All testes were determined using commercial kits (Spectrum, ELITech, BioSystems and Biomed Companies, Egypt) in accordance with the manufacturer's instructions.

Determination of lysozyme activity

Estimation of lysozyme activity by agarose gel lysis assay in serum sample (Schltz 1987).

Determination of Digestive Enzymes activities

To assess the effect of chlorella extract on digestive enzymes, 9 fish from each group (3 fish per aquarium) were were taken two times; at the day 40 of the feeding trial and post-challenge (at the end of the experiment, in the 55th day). Fish samples were anesthetized with eugenol (1: 10,000; Shanghai Reagent Corp., Shanghai, China), tissues including intestine and hepatopancreas were immediately dissected, the intestine was directly emptied and washed with ice-cold phosphate buffer (pH 7.0, 200 mM) for three times. Then the intestine and hepatopancreas were excised and homogenized using an electric homogenizer (XHF-D, Xinzhi, China) and the homogenate was centrifuged under cooling for 5 min at 5000 rpm. The supernatant was collected for assaying lipase and amylase activities according to the methods of Moss and Henderson (1999) using kits supplied by Spectrum (Cairo, Egypt) and Biodiagnostic (Giza, Egypt), respectively.

Water quality parameters  

      During the experiment, the mean values of water quality parameter (±SD) were as follows: water temperature 25 ± 2^°C; dissolved oxygen 6.5 ± 0.5 mg/L; pH 7.5 ± 0.8; total ammonia 0.021 ± 0.015 mg/L; nitrite 0.022±0.01 mg/L; and nitrate 0.8 ±0.2 mg/L, All water quality parameters were within the acceptable ranges (Abdelhakim et al. 2002).

Statistical analysis

The quantitative data was evaluated using one-way variance analysis (ANOVA). All data were analyzed statistically using the SPSS (version 21) software. The mean level was p<0.05, and the results are analyzed as means±standard error (SE).

Results

Antimicrobial assays

The result of antibacterial susceptibility test showed that the present isolate of E. tarda was sensitive only to Oxytetracycline and Gentamicin, and resistant for others. Oxytetracycline application is more suitable treatment in fish than Gentamicin (need injection). Chlorella extract Showed distinct antibacterial activity against E. tarda with 11.5 ±0.3 mm inhibitory zone. Minimum inhibitory concentration were 3.125 mg/ ml. The inhibitory zone of oxytetracycline and Gentamicin were 18.3±0.32 and 17.4 ±0.20 mm respectively.

Clinical signs

Non infected carps of Gp1 were healthy without any apparent lesions or mortalities and behaved normal upto the end of the experiment

 While carps of Gp2 which infected experimentally with E. tarda showed external and internal signs of infection started at the 5th day post-challenge, which are similar to those of natural infection as shown in Figure 1. These symptoms were, lethargy, off food, ascites and skin erosions, protruded hemorrhagic anal opening, several skin hemorrhagic patches and loss of colouration with detachment of scales. Internally there were liver enlargement with congestion or paleness, congestion and enlargement of kidney and internal organs. The number of carps which exhibited external signs of infection at the supplemented groups (Gp3 and Gp4), were much less than the control (Gp2), also the signs that appeared on them were less severe (including, anorexia, loss of reflexes, slight hemorrhagic patches and erosions on skin). Re-isolation of E. tarda occured from the spleen, kidney, heart and liver of challenged fish directly after symptoms appearance.

Figure 1.(a-i): Experimentally infected common carp fish with E. tarda showing a,b ascites and skin erosions. c. Protruded hemorrhagic anal opening. d, skin hemorrhagic patches. e, loss of colouration with detachment of scales. f,g liver enlargement with congestion or paleness. h,i congestion and enlargement of kidney and internal organs.

Growth performance and survival rates

As shown in Table 1, there were a dose-dependent significant increase in levels of growth indices (weight gain rate and SGR) with significant decrease in FRC were obtained in chlorella supplemented groups than the control, at both days 20 and 40 of the experiment, with special attention that the indices were improved proportionally with increasing the dose and the duration of supplementation of chlorella, no significant difference was observed in the growth indices of Gp1, Gp2 and Gp5. The carp’s survival percent challenged with E. tarda was elevated from 53.3% at Gp2 up to 90, 86.6 and 80% at Gp5, Gp4 and Gp3 respectively. This means that chlorella supplemented groups gave a satisfactory survival rates compared to the use of antibiotics and its hazards.

Table (1) effect of chlorella vulgaris extract on growth performance and survival rates (Mean ± SE).

The various letters in same raw indicate statically significant differences when (P< 0.05). Gp1 negative control, Gp2 positive control, Gp3(0.1 g chlorella extract), Gp4 (0.2g chlorella extract), Gp5(Oxytetracycline).

Serum biochemical parameters

As indicated at Table 2, after 40 days from chlorella supplementation, the levels of albumin, globulin and total protein were significantly elevated in the supplemented groups than control group (Gp1), especially Gp4. Post challenge their levels clearly decreased at Gp2 followed by Gp5, while Gp4 and Gp3 not significantly affected, as there's still an increase in their levels compared to the control. Albumin/Globulin (A/G) ratio recorded no significant difference in its ratio in all groups.

Gp 4 and 3 which supplemented with chlorella extract for about 40 days exhibit a normal liver and kidney function with a normal level of ALT and AST, ALP, creatinine and urea as the control (Gp1). While after challenge there were an elevation in their activities in all challenged groups especially Gp2, followed by Gp5 which treated with oxytetracycline, however chlorella supplemented groups exhibit significant lowered levels.

Table (2): Effect of chlorella vulgaris extract on some of serum biochemical parameters (Mean ± SE).

The various letters in same raw indicate statically significant differences when (P< 0.05). P.ch. (post challenge), Gp1 negative control, Gp2 positive control, Gp3(0.1 g chlorella extract), Gp4 (0.2g chlorella extract), Gp5(Oxytetracycline).

lysozymal activities

The present evaluation revealed an elevated lysozymal activities in the supplemented groups with chlorella over the time of the experiment. post challenge all groups had elevation in its activity in comparison with the control as shown in Table 3, the supplemented groups with chlorella exhibit a significant increase in lysozymal activity than the group which treated with oxytetracycline.

Digestive Enzymes activities

Table 3 summarized the results of amylase and lipase activities, at the day 40 of the experiment, there were an elevation in their activities in chlorella supplemented groups (Gp4 and Gp3) than control and this elevation was increased with increasing the concentration and the duration of supplementation upto 40 days. Post challenge, the levels of amylase were arranged descendingly as follow Gp1, Gp4, Gp3, Gp5 then Gp2, with the knowledge that, lipase activity not significantly affected with the infection only at chlorella supplemented groups particularly Gp4.

Table (3): Effect of chlorella vulgaris extract on lysozymal, Amylase and lipase activities (Mean ± SE).

The various letters in same raw indicate statically significant differences when (P< 0.05). P.ch. (post challenge), Gp1 negative control, Gp2 positive control, Gp3(0.1 g chlorella extract), Gp4 (0.2g chlorella extract), Gp5(Oxytetracycline).

Discussion

The incorrect and excessive use of chemicals and antibiotics against various bacterial diseases in fish farming possess a distinct threat to the growth and development of a sustainable and successful fish farming industry (Bondad-Reantaso et al. 2023). Therefore, it was necessary to search for an alternative and safe methods to the use of antibiotics, which in turn enhance the fish immunity and resistance to various diseases.

Recently, there has been increased demand for the use of microalgae in fish farms as a complementary nutritional additive. Chlorella vulgaris extensively applied in fish farming as a probiotic supplement (Abdelhamid et al. 2020). Chlorella contain phytonutrient called Chlorella growth factor (CGF), which is rich in nucleic acid-related substances such as peptides, proteins, amino acids, vitamins, vital sugars, minerals, a significant concentration of lipids; polysaccharides, pigments, and certain bioactive components which involved in many physiological activities (Ajiboye et al. 2012; Khani et al. 2017).

 The current evaluation was done for determination of the efficacy of chlorella vulgaris extract supplementation on the various indices of growth performance, some biochemical parameters of blood, lysozymal activity, amylase and lipase enzyme activities as well as survivability of common carp fish infected with E. tarda challenge.

The current findings of antimicrobial assays revealed that chlorella extract showed distinct antibacterial activity against E. tarda with 11.5 ±0.3 mm inhibitory zone and MIC were 3.125 mg/ ml. As Chlorella species found to contain a potent antibacterial compound called chlorellin (Mostafa 2012).

Also the present evaluation revealed an improvement in all growth indices of carps which supplemented with chlorella extract especially with the concentration of 0.2g/ kg diet (Gp4), this improvement significantly elevated also with long-term feeding of the extract upto 40 days. This result agreed with various reports which indicated that chlorella powder supplementation induce the growth performance and feed utilization in juvenile Japanese flounder Paralichthys olivaceus. (Kim et al. 2007). Common carp fish (khani 2017; Abdulrahman et al. 2019). African Catfish (Clarias Gariepinus) (Enyidi 2017). Oreochromis niloticus fingerlings (Abdel-Tawwab 2022). And L. rohita fingerlings (Pradhan et al. 2023).

Pakravan et al. (2018) stated that the growth enhancement associated with chlorella powder supplementation might be attributed to growth factors like adequate macronutrient volume and bioactive substances. Also, it may be due to the high digestibility of the microalgae (Khani et al. 2017).

Digestive enzymes activity assay is a reliable method that can be used as acode for the digestive processes and nutritional status of fish (Abolfathi et al. 2012).  In the present investigation, we found that chlorella supplementation could significantly elevate the activity of amylase and lipase of common carp after 40 days of supplementation, however post challenge there were a decrease in the activity of amylase in all groups than the control. The enhancement of the activity of these enzymes enhanced the digestion and feed utilization rate and so the rate of growth. Similar results were obtained by Xu et al. (2014) who claimed that C. vulgaris could increase digestive enzymes and results in improving growth performance and immune response. Also, Radhakrishnan et al. (2015) reported that, the growth and level of digestive enzymatic activities of M. rosenbergii fed C. vulgaris meal up to 50% level increased significantly. Similarly, khani et al. (2017) who recorded that chlorella could significantly increase the digestive enzyme in the hepatopancreas and intestine of koi carp.

Chlorella vulgaris possesses immune-stimulatory enhancement properties, aids pigmentation and digestion, and has scavenging and peroxidation properties (Khani et al. 2017; Zhang et al. 2014).

Furthermore, it was found that the levels of TP, albumin and globulin were elevated after supplementation of chlorella extract throughout the experimental period particularly Gp4. Post challenge (after 55 days), their levels clearly decreased at Gp2 followed by Gp5. A similar outcome was recorded in various reports after supplementation of chlorella powder for Gibel carp (Xu et al. 2014) and L. rohita fingerlings (Pradhan et al. 2023).

Albumin synthtization occurred in liver and act as transporter for protein (Anderson et al. 1979). So, the high level of TP and albumin which resulted from chlorella supplementation is an indication for liver function enhancement (khani et al. 2017).  Moreover, Globulins play a distinct role in the immune response of fish (Kumar et al. 2013). So, a strong innate immunity in fish is associated with the enhancement of the content of TP, albumin, and globulin content (Wiegertjes at al. 1996; Yildiz et al. 2002; Yu et al. 2006; khani et al. 2017).

Furthermore Albumin/Globulin (A/G) ratio in the current evaluation, recorded no significant difference in its ratio in all groups. In mammals A/G ratio is used for assessment of renal and hepatic dysfunction, and so the unchanged A/G ratio after supplementation of chlorella is an indication for the healthy status of supplemented fish (Pradhan et al. 2023).

Furthermore, the renal and hepatic function biomarkers in the present investigation revealed that, chlorella supplemented groups for about 40 days exhibit a normal level of ALT and AST, ALP, creatinine and urea as the control one, so its supplementation didn't induce any adverse effects on liver and kidney. After challenge there were an elevation in their activities in all challenged groups especially Gp2, followed by Gp5 which treated with oxytetracycline, however chlorella supplemented groups exhibit significant lowered levels.  A similar record was obtained by Fadl et al. (2017); Khani et al. (2017); Abbas et al. (2020); and Abdelhamid et al. (2020) after chlorella powder supplementation.

Also, Zahran et al. (2019) found that fish diet supplemented with powder of chlorella, improve the bioactivity of kidney and altered the damage which was caused by sodium arsenate toxicity in Oreochromis niloticus fish.

These outcomes may be attributed to the antioxidant activity and a hepatoprotective properties of chlorella (Goiris et al. 2012), and its supplementation protect the cell wall of the hepatic cells form the free radicals oxidative damage which induced during bacterial infection and prevent their enzymes from leaking into the blood circulation during infection. Moreover, Radhakrishnan et al. (2014) observed high concentration of vitamin E and C in the hepato-pancreas and musculature of M. rosenbergi fed a chlorella vulgaris supplement which has antioxidant activity and so causing protection to the integrity of cells.  As well as the high concentration of carotenoids, espcially astaxanthin in C. vulgaris which protect the parenchyma of kidney from oxidation by scavenging free radicals and suppressing oxidative stress damage (Pakravan et al. 2018).

The response of innate immunity is the first defence of fish against microbial infections. The present evaluation revealed an elevated lysozymal activities in the supplemented groups with chlorella extract over the time of the experiment. The supplemented groups (Gp3 and Gp4), exhibit a significant increase in lysozymal activity than the group which treated with oxytetracycline. Similar results were recorded by Zahran and Risha (2014); Xu et al. (2014); El-Habashi et al. (2019); Abdel-Tawwabet al. (2022) and Pradhan et al. (2023) with supplementation of chlorella powder. Also, Govindharajan and vairakannu (2021) found that serum lysozymal activity increase considerably in the groups treated with mixed algal extract including chlorella vulgaris in common carp fish.

These findings might attribute not only to the stimulatory effect of Chlorella to increase number and activity of phagocytic cells specially macrophages that enhance innate response via macrophage activation (Liu et al. 2006), but also to its high content of natural antioxidants such as chlorophyll, polyphenol, vitamins, sulphur-containing compounds that have the capacity to scavenge free radicals (Abdel-Tawwab et al. 2022; Aly et al. 2022).

Also, our findings recorded that carp’s survival percent challenged with E. tarda was elevated from 53.3% at Gp2 up to 90, 86.6 and 80% at Gp5, Gp4 and Gp3 respectively. This means that chlorella supplemented groups gave a satisfactory survival rates compared to the use of antibiotics and its hazards.

 These findings are in agreed with the results of Khani (2017) and Lim et al. (2018) who indicated that the carotenoid content enhances growth performance, stress tolerance, survival, physiology, resistance of disease as it protects host cells from oxidative damage caused by pathogenic microbes and immune status in several animal species. Moreover, Govindharajan and vairakannu (2021) stated that mixed algal extract supplementation including chlorella induced a high sustainability, immune response and survivability against A. hydrophila infection in common carp. Also, Dinev et al. (2021) stated that green microalgae as chlorella Vulgaris are having the ability of producing a wide range of active substances with antioxidant activity, immunostimulant, cytotoxic and antimicrobial that improve health status and increase resistance of disease.

Conclusions

The outcomes of this evaluation indicated that fish which fed a prepared experimental diet incorporated with Chlorella vulgaris extract with concentrations 0.1 and 0.2g /kg diet resulted in improving growth performance, digestibility, immunity and survivability, without any adverse effect on liver and kidney function, so chlorella vulgaris extract is considered an optimal choice as a safe dietry supplement enhancing performance and survivability of fish.