Levamisole Enhances Growth, Haemato-biochemical Indicators, Resistance to Aeromonas veronii and Expression of Immune Genes in Asian Seabass, Lates calcarifer

Diseases are the primary limiting factors for the development and expansion of aquaculture (Ringo et al., 2010). Traditionally, control and prevention of diseases were achieved using a wide range of antibiotics, pesticides, disinfectants and other chemicals. The use of chemotherapeutic drugs and antibiotics for treating illnesses has drawn a lot of criticism because of their unfavorable side effects, including residue buildup in tissue and the environment, the emergence of drug-resistant bacteria, immunosuppression and decreased customer demand for fish as a food source (Yasin et al., 2023). Consequently, novel preventative approaches need to be developed to effectively address both emerging and established diseases (Ringo et al., 2010). Immunostimulants serve as dietary supplements that enhance resistance to specific infections and activate the fish innate defense mechanisms. Levamisole is a synthetic anthelmintic used in mammals which also has the capability to elicit innate-specific immunity response in fishes (Reverter et al., 2014). Levamisole has the capacity to stimulate the synthesis of interferon (IFN) and IL-6 through enhanced expression of MHC receptors (Holcombe et al., 2006). Several studies demonstrated that levamisole boosted immunity by mimicking the action of the thymic hormone thymopoietin (TMPO) which is identified as a biologically active peptide comprising 49 amino acids and located within the thymus (Weber et al., 1999). Hence levamisole has the potential to adopt a tertiary structure resembling thymopoietin and stimulate lymphocytes through its imidazole component also reported as a growth promotor in carp (Baba et al., 1993; Gopalakannan and Arul, 2006), nile tilapia (Bedasso, 2017) and rainbow trout (Kajita et al., 1990). Asian seabass (Lates calcarifer) is a commercially important species, known as barramundi, is widely cultured in Southeast Asia and Australia using fresh, brackish and marine water resources (Glencross et al., 2016). Similar to many other aquaculture species, the adoption of intensive farming practices has led to a rise in the occurrence of bacterial, viral and parasitic infections in L. calcarifer farming (Anderson and Norton, 1991; Azad et al., 2004; Kumar et al., 2007). Aeromonas veronii stands out as a prevalent pathogen in aquaculture, with the ability to affect various aquatic species. Research has demonstrated that A. veronii exhibits a higher level of virulence compared to Aeromonas hydrophila. Consequently, this triggers the activation of the aer gene, leading to enhanced bacterial adhesion ability within host cells. Moreover, the quorum-sensing mechanism significantly contributes to the infection process of A. veronii in seabass. Lateolabrax maculatus infected with A. veronii exhibit acute mortality, marked by ulcerations on the body surface and congestion, as well as hemorrhaging in internal organs like the liver, kidney and spleen (Wang et al., 2021). Keeping this in view, this study was undertaken to evaluate the effect of dietary levamisole on the growth, haemato-biochemical parameters, disease resistance against Aeromonas veronii and immune-related gene expression in Asian seabass, Lates calcarifer.

Fish husbandry
 
Asian seabass (Lates calcarifer) seeds (2.4±0.3 cm; 1.2±0.2 g) were purchased from the hatchery located at Rajiv Gandhi Centre for Aquaculture in Sirkazhi, Tamil Nadu, India. The health status of the fish were examined immediately upon arrival to the experimental facility. The fishes were acclimatized for 30 days in Fiber-reinforced plastic (FRP) tanks (1000L capacity each) in a controlled laboratory environment, by feeding a diet with 45% crude protein.
 
Experimental diet preparation
 
A basal feed with 45% crude protein and 10% crude lipid was prepared. Levamisole (Sigma Aldrich) was incorporated into the basal diet at varying levels, viz., 0.0 mg/Kg (C), 75 mg/Kg (LVT1), 150 mg/Kg (LVT2), 300 mg/Kg (LVT3) and 600 mg/Kg (LVT4) based on the methods of Li et al., (2006); Kumari and Sahoo (2006); Lim et al., (2019) and Pahor-Filho et al., (2017). Briefly, the diets were prepared by mixing the ingredients (Table 1). Preparation of dough by adding water and grinding in a meat grinder followed by steam cooking for 20 min and pelletizing through 2-mm dia die in a sinking feed pelletizer (Jinan Sunpring, China). The feeds were stored in airtight containers at room temperature for later use (Prabu et al., 2021).
 
 
 
Experimental setup
 
For the experimental trial, fish with an average weight of 3.29±0.4 g were segregated into 15 groups with triplicates for the control and four treatment groups (C, LVT1, LVT2, LVT3 and LVT4) with 20 fish each. Subsequently, these fish were transferred to rectangular FRP tanks (250 L capacity) provided with ample aeration. During the 60-day experimental trial, the fish were fed daily with four feeding sessions (at 8:00 am, 12:00 pm, 4:00 pm and 8:00 pm). The values of water quality parameters viz., water temperature (28±1°C); dissolved oxygen (6.6±0.4 mg/L); pH (7.8±0.2), salinity (0 ppt), hardness (132±3 ppm) and alkalinity (97±2 ppm) (Boyd, 2012) were recorded daily and maintained at optimal ranges.
 
Fish growth and feed utilization
 
After the feeding trial, fish specimens were chosen randomly for comprehensive evaluation of growth performance, haemato-biochemical indices and analysis of growth and immune gene expressions related to both growth and immune functions. Biogrowth parameters such as the feed conversion ratio (FCR), weight gain (g), survival rate (%) and protein efficiency ratio (PER) were also recorded (Abdel-Tawwab et al., 2010).
 
Hemato-biochemical indicators
 
After the 60-day experimental feeding trial, the serum was collected from the experimental fishes by anesthetizing with 100 mg/L of MS-222 (Sigma-Aldrich, USA). The blood samples were collected using a 1 ml syringe by kept at the ambient temperature for 1h and at 4°C for 4 h followed by centrifugation (4°C, with 1500 × g for 5 min). The supernatant was collected and stored at -20°C until needed. Neubauer-hemocytometer was used to count the erythrocytes (Ery) and WBC followed by hemoglobin (Hb [g/dl]) level (Erdal et al., 1991). Blood indices, including mean corpuscular hemoglobin concentration (MCHC [g/dl]), mean corpuscular hemoglobin (MCH [pg]) and mean corpuscular volume (MCV [fl]) were also assessed (Wintrobe, 1934). The values of glucose (GLU) (Sigma Diagnostics, India), Serum cholesterol (CHO) (Parekh and Jung, 1970), Triglycerides (TG) (Rice, 1970) and serum total protein (Reinhold, 1953) were also analyzed.
 
Bacterial pathogen
 
A. veronii (CAK4/SRLAAH/2022) (Gen Bank Acc no. OP752155) isolated from an infected Asian seabass, identified and maintained at the microbial archive of State Referral Laboratory for Aquatic Animal Health, TNJFU- Madhavaram campus was used for the experimental challenge in this study. The isolate was revived by growing for 24 h at 30°C in nutrient broth (HiMedia). Growth was assessed in a spectrophotometer at OD 600 and centrifuged at 3000 × g for 15 min. Bacterial pellets were obtained, dissolved and vortexed in sterile 0.15 M physiological buffer saline (PBS) solution and preserved by storing them in tryptic soy broth (TSB) supplemented with 15% (v/v) glycerol and this was kept at a temperature of -70°C.
 
Experimental bacterial challenge
 
After the 60-day feeding trial, ten fish from each of the groups were experimentally challenged by intraperitoneal injection with A.veronii  0.1mL at 2.5 × 10⁴ cells/ml estimated by the LD50 method in the laboratory. The fish were observed for 15 days for the clinical signs and mortality. Reisolation of the pathogen was carried out on Aeromonas Isolation Medium Base (HIMEDIA). To confirm the identity of the bacterial pathogen, 16S rRNA PCR and subsequent sequence analysis were performed (Weisburg et al., 1991).
 
Quantitative real-time PCR (qRT-PCR)
 
Total RNA was extracted from aseptically collected fish muscle and kidney tissues using the RNA iso-plus kit (Takara Bio Inc. Japan), following the manufacturer’s provided guidelines. Subsequently, 2 µg of total RNA was utilized to synthesize the first-strand complementary DNA (cDNA), following the manufacturer’s instructions (Thermo Scientific, USA). The relative expressions of Hsp70 and Hsp90 were assessed following the procedures described by Glencross et al., (2016) and Fu et al., (2021) respectively. The GH and MSTN expressions were assessed by the methodology of Ezhimathi et al., (2022). The methodologies of Korni et al., (2021) and Hsieh et al., (2010) were employed to analyze TLR2 and TLR4 expression. The assessment of the ß-actin transcript was used as an internal control (Wahyudi et al., 2018). The details of primers, base pair and protocols are presented in Table 2.
 

       
PCR amplification was conducted using the C1000 Touch thermal cycler with the CFX96 Real-time PCR system (Bio-Rad, USA). For qPCR, the reaction mixture comprised approximately 20ng of cDNA template, 10 µM each of forward and reverse primers and 1x SYBR Green PCR Master Mix Kit (Takara Bio Inc, Japan), along with 20 µl of nuclease-free water. The qPCR cycle threshold (Ct) values were used to calculate the relative gene expression level displayed as 2^-ΔΔCt (Livak and Schmittgen, 2001).
 
Statistical analysis
 
To assess the significant differences between the means, one-way analysis of variance (ANOVA) followed by Tukey’s multiple range tests using IBM-SPSS Statistics version 20 were performed. The data are presented with mean values and their respective standard deviations (SD). A significance level of p <0.05 was applied to identify statistically significant differences among the treatment groups.

Growth performances and feed utilization
 
Levamisole supplemented diet fed groups showed significant (p<0.05) improvement in feed efficiency and growth performance in L.calcarifer (Table 3). Fish fed with LVT3 diet exhibited significantly (p<0.05) higher final body weight, improved feed efficiency ratio and lesser feed conversion ratio compared to control and other dietary groups were shown in Fig 1A, B and C. Similarly, levamisole-fed Cyprinus carpio displayed a lower feed conversion ratio and a higher specific growth rate (Maqsood et al., 2009). Caspian brown trout (Salmo caspius), juvenile Piaractus mesopotamicus pacu fish and beluga fish that were fed with levamisole supplemented diet showed an improved protein efficiency ratio (Yuji Sado et al., 2010; Eslami and Bahrekazemi, 2019). Several studies have documented the immune-enhancing properties and growth-promoting effects of levamisole in various fish species viz., Cyprinus carpio, angelfish (Pterophyllum scalare), gilthead seabream (Sparus aurata L.) and nile tilapia (Oreochromis niloticus) (Kajita et al., 1990; Maqsood et al., 2009; Mulero et al., 1998; Gopalakannan and Arul, 2006; Kasiri et al., 2011; Bedasso, 2017). 
 

 
Haemato-biochemical parameters
 
In comparison with the control group, the LVT3 fish group showed increased levels of hemoglobin (Hb), packed cell volume (PCV), erythrocyte count, mean corpuscular hemoglobin concentration (MCHC), cholesterol, triglycerides, lymphocytes, total protein, albumin and globulin. Experimental challenge with A. veronii resulted in a significant variation (p<0.05) in the haemato-biochemical parameters such as increased glucose level and lowest level of packed cell volume (PCV), hemoglobin (Hb), erythrocyte count (Ery), mean corpuscular hemoglobin concentration (MCHC), triglycerides (TG), total protein, albumin and globulin in the control group. Whereas levamisole-supplemented diet-fed LVT3 groups did not show significant variation compared to other treatments after experimental infection with A. veronii depicted in Table 4,5. After the experimental challenge, a notable rise in WBC counts and levels of polymorphs, lymphocytes, eosinophils and monocytes, was noted in the LVT3 group. Similarly, enhancements in non-specific immune responses, including increased counts of “natural killer” cells and heightened phagocytic activity were observed in levamisole-injected Oncorhynchus mykiss after challenge with virulent Vibrio anguillarum (Kajita et al., 1990). According to Kowalska et al., (2015) and Wijendra et al., (2007), the addition of levamisole to the diet of pikeperch (Sander lucioperca) and Labeo rohita respectively, resulted in a significant increase in hematological indices such as hemoglobin, red blood cell (RBC) and white blood cell (WBC) counts.
 

 

 
Disease resistance
 
The LVT3 group exhibited the lowest cumulative mortality 13.34% when challenged with A. veronii whereas the LVT2 group showed a 20% mortality rate. The fish fed with 600 mg/Kg (LVT4) levamisole in their diet had a mortality rate of 33.3% indicating higher concentrations of levamisole caused agranulocytosis and leukopenia in fishes (Midthun et al., 2021) whereas the low concentration of levamisole 75 mg/Kg (LVT1) caused 26.67% mortality when challenged with A. veronii. Fishes fed with the diet (C) showed significantly (p<0.05) higher mortality (53.34%) (Fig 1D). Extensive research on feeding levamisole in some fishes showed that it improves the functioning of certain components of the innate immune system as observed in the present study (Kajita et al., 1990; Siwicki et al., 1989; Mulero et al., 1998).
 

 
Growth gene and immune-related gene expression
 
The relative expression of growth genes (GH, MSTN), stress genes (Hsp90, Hsp70) and immune genes (TLR2, TLR4) in juvenile L. calcarifer fed diets containing levamisole supplementation showed in Fig 2. Fish raised in different treatments (LVT1, LVT2, LVT3, LVT4 and control) showed a distinct pattern of significant (p<0.05) expression. The relative expression of the GH (Growth Hormone) gene was up-regulated in the LVT3 group 4.46 fold (p<0.05) compared to the control (C) whereas, 2.54-fold in LVT1, 3.47-fold in LVT4 and 4.10-fold in LVT2 were recorded. Nevertheless, when different levels of dietary levamisole were administered, there were no notable changes observed in myostatin (MSTN) mRNA expression within the muscle tissues of L. calcarifer. Earlier studies have shown that dietary supplementation of olive leaf extract enhances GH gene expression in common carp Cyprinus carpio (Zemheri-Navruz et al., 2020). The expression levels of Hsp70 and Hsp90 exhibited a significant downregulation, with a fold change of 0.04 in the LVT3 group after being infected with A. veronii when compared to the control group. According to Hassaan et al., (2021), Hsp70 gene expression was significantly reduced (p<0.05) when fish were fed with ß-carotene and phycocyanin in their diet. In the current research, it was observed that the addition of levamisole LVT3 (300 mg/Kg) to the diet of L. calcarifer decreased the mRNA expression levels of Hsp90. Tan et al., (2017) demonstrated that the mRNA expression levels of the Hsp90 gene were reduced in golden pompano when fed with hawthorn extract. This downregulation of Hsp90 expression was likely a result of the increased tolerance of the fish towards common stresses. The relative expression of TLR2 and TLR4 genes was upregulated in the LVT3 group with 4.94 and 4.90 fold (p<0.05), respectively. Upregulation of the TLR2 has been observed in the antimicrobial peptide Epinecidin-1-expressing Artemia cyst-fed Nile tilapia (Ting et al., 2018). TLR4 plays a major role in maintaining the immune system of animals, particularly in the gut (Cario and Podolsky, 2000). Previously, TLR4 upregulation had been documented in transgenic zebrafish following infection with Vibrio vulnificus (Hsieh et al., 2010). In the present study, LVT3 showed decreased stress gene expressions and enhanced immune gene expressions. Relative higher expression of the immune gene indicates, enhanced pathogen defense, improved clearance of infected cells and reduced disease severity (Mohanty and Sahoo, 2010) in levamisole diet-fed fishes after experimental infection with A. veronii, as a result, the mortality was lesser in the LVT3 group.
 

Levamisole shows great promise as an immunostimulant in L. calcarifer. In this study, dietary supplementation with levamisole exhibited improved growth performance, feed utilization and disease resistance against A. veronii in L. calcarifer. When compared to other dietary treatments and the control group, fish that were fed diets supplemented with 300 mg/Kg levamisole demonstrated superior performance in various aspects viz., enhanced growth, improved disease resistance, upregulated expression of growth and immune-related genes and higher survival rates when exposed to A. veronii infection. In conclusion, the findings of this study suggest that levamisole is a promising immunostimulant to be used for enhancing the growth and resistance to A.veronii infection of L. calcarifer.

The authors sincerely thank Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, Tamil Nadu, India for the grants and offering the research facilities of the State Referral Laboratory for Aquatic Animal Health, TNJFU- Madhavaram campus.

The authors have no conflicts of interest to declare.