Immunomodulatory effects of Carica papaya leaf powder in the diet of Labeo catla challenged with Aeromonas hydrophila

Introduction

Aquaculture plays a crucial role in global food security and economic development. According to FAO reports, the combined global production of fisheries and aquaculture reached approximately 223.2 million tonnes in 2022, with aquaculture alone contributing about 130.9 million tonnes (FAO, 2024). With the world population projected to reach nearly 10 billion by 2050, the demand for high-quality protein sources is expected to rise substantially. In this context, aquaculture has significant potential to expand aquatic food production and meet the increasing nutritional requirements of a growing and more affluent population. Consequently, aquaculture is particularly important for strengthening food security, especially in low-income and food-deficient regions (Khan et al., 2023; Hassan et al., 2025) [18,16] . Over the past few decades, aquaculture production has grown rapidly, and continued expansion is considered essential to satisfy future food demands (Rajabiesterabadi et al., 2020; Mabrouk et al., 2025) [29,20] . The intensification of aquaculture systems-characterized by high stocking densities, monoculture practices, and confined production environments-often creates favorable conditions for the rapid transmission of pathogens. Stress factors such as overcrowding, poor water quality, and inadequate nutrition can weaken the immune responses of fish, increasing their susceptibility to infectious diseases (Awad & Awaad, 2017; Quesada et al., 2013; Reverter et al., 2014) [4,27,30] . Despite significant progress in the farming of Indian major carps, several environmental and economic challenges continue to threaten the sustainability of the sector (Barrett et al., 2019) [7] .

In many Asian aquaculture systems, diseases frequently arise from primary or opportunistic pathogens that proliferate under stressful environmental conditions (Lategan & Gibson, 2003; Fang et al., 2004) [9,14] . Traditionally, antibiotics and synthetic chemicals have been used to prevent and control disease outbreaks. However, their extensive application has led to the emergence of antimicrobial resistance and the accumulation of chemical residues in fish tissues, raising concerns regarding food safety and public health (Okocha et al., 2018; Imtiaz et al., 2024) [23,17] . Among the bacterial pathogens affecting aquaculture, Aeromonas hydrophila is a facultatively anaerobic, opportunistic, rod-shaped, non-spore-forming bacterium belonging to the family Aeromonadaceae. It is widely distributed in aquatic environments and is capable of infecting a broad range of hosts, including freshwater and marine organisms. This bacterium has been extensively investigated due to its occurrence in water, food, and estuarine environments, as well as its antibiotic resistance and pathogenic potential (Odeyemi et al., 2012; Radu et al., 2003) [22,28] . Infections caused by A. hydrophila are responsible for severe diseases in fish, including dropsy, epizootic ulcerative syndrome (EUS), and ocular lesions (Roque et al., 2009) [31] . Consequently, there is increasing interest in alternative strategies that can enhance disease resistance while reducing reliance on antibiotics and improving feed utilization efficiency (Du et al., 2022) [11] . Various natural and biological approaches, including probiotics, plant-derived compounds, prebiotics, and synbiotics, have shown promising results in aquaculture due to their environmental safety, sustainability, and cost-effectiveness (Mabrouk et al., 2025; Aboleila et al., 2022) [20,1] . Medicinal plants, in particular, are considered valuable due to their availability, long-term safety, and therapeutic efficacy (Yilmaz et al., 2024). More than 60 plant species have been evaluated for their ability to enhance fish health and control diseases in aquaculture systems (Bulfon et al., 2015) [8] . Carica papaya L. (papaya), a member of the family Caricaceae, is widely cultivated throughout Asia. Numerous investigations have explored the biological activities of different parts of the plant, including fruits, leaves, seeds, shoots, roots, and latex (Otsuki et al., 2010) [25] . Papaya leaves, often regarded as agricultural by-products, contain a variety of bioactive compounds with significant pharmacological potential. These leaves exhibit immunomodulatory (Alhassan et al., 2024) [3] , antioxidant (Sharma et al., 2022) [3] , and anti-inflammatory (Cao et al., 2024) properties due to the presence of phenolic compounds (Espadas et al., 2019) [12] , flavonoids (Nugroho et al., 2017) [21] , and enzymes such as papain (Babalola et al., 2023) [5] . Papain is a cysteine protease capable of breaking down complex proteins into smaller peptides and amino acids, thereby facilitating digestion. Additionally, papaya leaves contain several bioactive constituents, including alkaloids, flavonoids, phenolic acids, and papain, which collectively contribute to their broad therapeutic potential (Palanisamy & Basalingappa, 2020) [26] . These compounds have also been reported to enhance antioxidant activity and reduce lipid peroxidation in biological systems (Otsuki et al., 2010) [25] . Therefore, the present study aimed to evaluate the effect of dietary supplementation with Carica papaya leaf powder on the immune response of Labeo catla when challenged with Aeromonas hydrophila.

Materials and Methods Experimental Design and Fish

The feeding trial was conducted for a period of 60 days using twenty experimental tanks, each supplied with adequate aeration to maintain appropriate dissolved oxygen levels. Prior to the experiment, all tanks were thoroughly washed and disinfected with potassium permanganate (KMnO₄) to ensure hygienic conditions. After disinfection, each tank was filled with approximately 40 L of filtered and disinfected freshwater. Fry of Labeo catla were obtained from Aqua-Fish Farm, Nal Sarovar (Nani Kathechi, Gujarat). The fry were transported to the laboratory in oxygenated polyethylene bags by road. Upon arrival, the fish were transferred to 500-L FRP tanks and acclimatized under continuous aeration. During acclimation, the fry were fed three times daily to facilitate adjustment to the new environment. Healthy individuals with an average body weight of 0.381 ± 0.001 g were selected for the feeding experiment. Five dietary treatments were prepared for the study: T 0 (control diet without Carica papaya leaf powder), T 1 (2.5%), T 2 (5%), T 3 (7.5%), and T 4 (10%) inclusion levels of dried papaya leaf powder in the formulated diets.

Collection of Carica papaya Leaves and Preparation of Leaf Powder

Leaves of Carica papaya were collected from a nearby farm and washed thoroughly with tap water to remove dust and impurities. The cleaned leaves were sun-dried for 3-4 days until completely dehydrated. The dried leaves were then ground into a fine powder using a grinder. The powdered material was sieved to obtain a uniform texture and subsequently stored in airtight polyethylene bags to preserve its quality until use.

Ingredient Proximate Analysis

Proximate composition of the feed ingredients was determined using standard analytical procedures described by AOAC (2000). The proximate composition of the ingredients is presented in Table 1.

Diet Preparation

Experimental diets were formulated using the Pearson square method. Five diets (T 0 -T 4 ) were prepared, where treatments T 1 , T 2 , T 3 , and T 4 contained varying levels of papaya leaf powder, while T 0 served as the control diet without papaya leaf powder. The required quantities of feed ingredients were weighed accurately according to the formulation described in Tables 2 and3. All ingredients were thoroughly mixed with an appropriate amount of water in a plastic tray to obtain a homogeneous mixture. The feed mixture was then subjected to steam cooking at 121 °C under a pressure of 15 lbs for 15 minutes. After steaming, vitamin and mineral premixes were incorporated into the mixture and mixed thoroughly. Subsequently, papaya leaf powder was added to the diets according to the treatment levels and mixed uniformly. The prepared feed mixture was pelletized using a mechanical pelletizer to produce pellets of uniform size and shape. The pellets were spread evenly on steel trays and sun-dried for 2-3 days until the moisture content was reduced to below 12%. The dried pellets were then stored in properly labelled airtight plastic containers until use.

Feeding

Fish were fed twice daily at 08:00 h and 17:00 h throughout the experimental period. Feed was provided at a rate of 10% of the fish body weight. The formulated diets contained approximately 30% crude protein. The feed quantity was adjusted every two weeks based on the growth performance of the fish to maintain an appropriate feeding regime.

Water Quality Management

Continuous aeration was maintained throughout the experiment to ensure adequate dissolved oxygen levels. The tanks were cleaned daily before feeding by siphoning to remove uneaten feed and accumulated waste materials. Approximately 10% of the tank water was replaced regularly with filtered and disinfected freshwater during the 60-day experimental period.

Minimum Inhibitory Concentration Test

At the end of the feeding trial, the immune response of Labeo catla fry fed papaya leaf powder was evaluated using Aeromonas hydrophila as the test pathogen. Bacterial isolates were first cultured in liquid medium and subsequently transferred onto nutrient agar plates. Blood samples were collected from the caudal peduncle of fish using a 1-mL syringe containing EDTA as an anticoagulant. The samples were transferred into Eppendorf tubes and centrifuged at 5000 rpm for 5 minutes.

Sterile paper discs were immersed in the supernatant for 15 minutes to ensure proper absorption. These discs were then placed on nutrient agar plates inoculated with Aeromonas hydrophila using sterile forceps. The plates were incubated at 35 °C for 72 hours. Zones of inhibition around the discs were measured and recorded after 24, 48, and 72 hours.

Effect of Papaya Leaf Powder on Disease Resistance of

Labeo catla Fry At the end of the feeding trial, a bacterial challenge test was conducted. Fish from each treatment group were injected intramuscularly with 0.1 mL of Aeromonas hydrophila suspension at a concentration of 1 × 10⁵ CFU/mL. The control group was injected with phosphate buffer solution (PBS). Ten fish from each replicate were selected for the challenge test. Following infection, fish survival was monitored for 10 days. During this period, fish were observed for clinical signs of disease and abnormal behaviour. Due to the small size of the fry, the commonly used intraperitoneal injection method was not feasible; therefore, the bacterial challenge was administered via intramuscular injection.

Statistical Analysis

The experimental data were analyzed using standard statistical procedures described by Snedecor and Cochran (1994) [35] .

Results and Discussion 1. Minimum Inhibitory Concentration (MIC) Test

The immune response of Labeo catla fry under different dietary treatments was assessed using the disc diffusion method, and the results are presented in Table 4.

Antibacterial activity was evaluated by measuring the diameter of the zone of inhibition at 24, 48, and 72 hours.

The observations revealed significant variations among the treatments at all sampling intervals. Furthermore, Figure 1 illustrates the differences in inhibition zones among the treatments after 48 hours of incubation. After 24 hours, the highest zone of inhibition was recorded in treatment T 4 (12.60 ± 0.24 mm), followed by T 3 ( Overall, the results clearly indicated that treatment T 4 produced the strongest antibacterial response, followed by T 3 , T 2 , T 1 , and T 0 . The progressive increase in inhibition zones over time indicates an enhancement in immune response due to the dietary inclusion of papaya leaf powder. In the present study, the incorporation of papaya leaf powder at 7.5% and 10% levels significantly improved the antibacterial activity of fish serum against Aeromonas hydrophila. These findings are consistent with the results reported by Ballad et al. (2011) [6] , who observed that papaya leaf extract at a concentration of 10 mg/ml produced an inhibition zone of 14.33 mm, indicating strong antibacterial activity. Similarly, Fakoya et al. (2019) [13] demonstrated that methanolic and ethanolic extracts of papaya leaves produced significant inhibition zones against A. hydrophila using the disc diffusion technique. The enhanced antibacterial activity observed in the present study may be attributed to the presence of bioactive phytochemicals in papaya leaves, including saponins, alkaloids, tannins, glycosides, and phenolic compounds, which are known for their antimicrobial, antibacterial, and antipathogenic properties ( [10,32,36] . Therefore, dietary supplementation with papaya leaf powder appears to strengthen the immune defense of fish and may serve as a natural immunostimulant in aquaculture systems.

Effect of Papaya Leaf Powder on Disease Resistance of Labeo catla Fry

Following the completion of the 60-day feeding trial, fish were subjected to a bacterial challenge test using Aeromonas hydrophila. Each fish received an intramuscular injection of 0.1 ml bacterial suspension containing 1 × 10⁵ cells/ml, and survival was monitored for 10 days. The survival data obtained after the challenge test are presented in Table 5.

The highest survival rate was recorded in T 4 (95.00 ± 5.77%), followed by T 3 (80.00 ± 8.16%), T 2 (67.50 ± 9.57%), T 1 (55.00 ± 12.91%), and T 0 (52.50 ± 5.00%). Statistical analysis indicated significant differences among the treatments (P<0.05), suggesting that dietary inclusion of papaya leaf powder significantly enhanced resistance against bacterial infection. [34] , who observed a 95% survival rate in Koi fish (Cyprinus rubrofuscus) when treated with papaya leaf extract against A. hydrophila. Likewise, Olusola and Nwokike (2018) [24] reported a survival rate of 83% in Clarias gariepinus when fish diets were supplemented with 3% papaya leaf and bitter leaf extracts, which improved resistance to A. hydrophila infection. The increased survival rates may be attributed to the antimicrobial and immunostimulatory properties of phytochemicals present in papaya leaves, such as saponins, alkaloids, tannins, glycosides, and phenolic compounds, which have been widely reported for their antibacterial and antipathogenic activities ( [32,36] . The present findings therefore suggest that dietary supplementation with papaya leaf powder, particularly at 7.5% (T 3 ) and 10% (T 4 ) levels, can significantly enhance disease resistance and immune response in Labeo catla fry. Such plant-based additives can serve as effective and ecofriendly alternatives to antibiotics in aquaculture, contributing to sustainable fish health management.

Physico-Chemical Water Parameters

The physico-chemical characteristics of the experimental water during the study period are presented in Table 6. Throughout the experimental duration, all water quality parameters remained within acceptable ranges suitable for the culture of Labeo catla fry. Water temperature varied between 24.2 °C and 29.7 °C across treatments. The pH ranged from 6.8 to 7.9, indicating slightly acidic to neutral conditions suitable for fish growth. Dissolved oxygen levels ranged from 5.4 to 6.6 ppm, which were adequate to support normal metabolic activity and fish health.

Total hardness values ranged from 140 to 230 ppm, while ammonia concentrations were maintained between 0.05 and 0.11 ppm, and nitrite levels ranged from 0.17 to 0.42 ppm. These values remained within permissible limits and did not exhibit significant variations among treatments. The stable water quality conditions observed during the experiment ensured that fish growth, immune response, and survival were primarily influenced by the dietary treatments rather than environmental stress factors.

Conclusion

The present study demonstrated that dietary supplementation with Carica papaya leaf powder significantly enhances the immune response and disease resistance of Labeo catla fry. Fish fed diets containing papaya leaf powder showed higher antibacterial activity against Aeromonas hydrophila, as evidenced by increased zones of inhibition in the disc diffusion assay. Among the treatments, the highest inclusion level (10%, T 4 ) produced the strongest immune response and the greatest inhibition zone at all observation intervals. Similarly, the challenge test revealed improved survival rates in fish receiving papaya-supplemented diets, with the highest survival recorded in T 4 (95%), followed by T 3 (80%). These findings indicate that papaya leaf powder acts as a natural immunostimulant, likely due to the presence of bioactive phytochemicals such as flavonoids, phenolic compounds, alkaloids, and tannins. Furthermore, water quality parameters remained within acceptable ranges throughout the experimental period, ensuring suitable rearing conditions. Overall, the results suggest that dietary inclusion of papaya leaf powder, particularly at 7.5-10%, can be an effective, eco-friendly alternative to antibiotics for improving fish health and disease resistance in aquaculture systems.

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