2.1. Chemicals:
2.1.1. Fipronil: Fipronil 20% was obtained from Yongnong Biosciences Co., Ltd. China. Technical grade Fipronil (C12H4Cl2F6N4OS) (99.1% pure) was manufactured by Bio Quest International Private Limited, Mumbai, India, and it consists of two isomers at a ratio of 50:50. Fipronil’ doses were estimated [26].
2.1.2. Lead: The solid white crystal form of lead nitrate Pb(NO3)2 was obtained from Fisher Scientific Company in Canada.
2.1.3. Beta glucan: β-1, 3-glucan (Sigma, U.S.A.) was dissolved in phosphate buffered saline (PBS), thoroughly mixed, and subsequently added to basic diet constituents at a rate of 0.1% before palletization. The feeding period and β-1,3-glucan dose used in this study were based on the earlier report [27].
2.1.4. Cytokines and antibodies: Fish Interleukin-1β (IL-1β), ELISA Kit, Cat. No. MBS700023 and Fish Interleukin 6, (IL-6) ELISA Kit, Cat. No. MBS015740 were used in this study for the detection of IL-1β and IL-6 levels. The kits were obtained from MyBiosource Co. (San Diego, California, USA). The Immunoglobulin M (IgM) ELISA Kit. Catalog No. CSB-E12045Fh, was purchased from CUSABIO BIOTECH CO., Ltd. and it was used to quantitatively determine the IgM level.
2.2. Fish management and maintenance
Two hundred forty Nile catfish with an initial 40 ± 3.0 g body weight (mean ± SD) and 14.71 ± 1.23 cm of fork length were used in this study, and they were obtained from a local hatchery (Abbasa, Ash Sharqiah, Egypt). The fish were transferred to the Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Zagazig University, Egypt, where the experiments were performed. Initially, fish were immersed in a salt bath of NaCl at a concentration of 2.5% for five minutes to get rid of external parasites and fungal infections. The fish were acclimatized for two weeks before starting the actual experiments, which lasted a total of two months. During the acclimatization period, fish were fed a commercial pelleted feed containing 32% protein and were placed in glass aquaria (80 x 40 x 30 cm capacity). Each aquarium had 60 litres of chlorine-free tap water. An aerator was used to provide air supply; a thermostatically controlled heater kept the water temperature at 26°C; the pH of the water was kept at 6.5-7.0; dissolved oxygen averaged 60.5 mg L-1; ammonia averaged 0.01 mg L-1; and nitrite was 0.20 mg L-1. These parameters were measured routinely with a freshwater kit (La Motte®, Chertestwon, MD, USA). Approximately 30% of the water was changed daily, and a constant water flow was maintained.
2.3. Experimental design
After the acclimatization period, the 240 catfish were divided into four experimental groups (60 fish per group, each group in triplicate) after acclimation and then placed into 16 aquaria at random (4 aquaria per group with 15 fish per aquarium). The first group served as a control, and the fish were fed a basic diet without any additions or treatments, while the fish in the second group were fed a basic diet supplemented with 0.1% of β-1, 3-glucan, and the fish in the third were fed a basic diet and exposed to a combination of lead nitrate at 0.041 mg/L (1/10 96 h LC50) according to the New Pesticide Fact Sheet (1996) and fipronil at 2.8 mg/l (1/10 96 h LC50) according to Mahmoud (2013). The fourth group of fish received a basic diet supplemented with 0.1% of β-1, 3-glucan before being exposed to lead nitrate and fipronil at the same mentioned concentrations. All of the fish in the various experimental groups were fed their individual diets ad libitum four times daily at a rate of 3% of their body weight. Every day, the fish were checked for any changes in their general health, clinical signs, odd behavior or coloration, or respiratory distress. The affected fish's post-mortem lesions and mortality rate were documented.
2.4. Molecular determination of IL-1β, IL-2, or IL-6
Spleens were obtained from dead or euthanized fish, quickly maintained in liquid nitrogen and stored at -80ᴼC to be used in a semi-quantitative RT-PCR [28] using the appropriate specific set of primers to measure IL-1, IL-2, or IL-6 cytokine gene expression (Table 1). RNA was extracted from various fish groups using the Gene JET RNA purification kit (Fermentas, UK) according to the manufacturer's instructions. A Nano-Drop ®ND-1000 Spectrophotometer (Wilmington, Delaware, USA) was used to verify the quantity and purity of the RNAs collected. The reverse transcriptase enzyme Superscript II RNase H (Invitrogen, Carlsbad, CA, USA) was used to make cDNA. To express arbitrary units of relative abundance, the values of the specific targets were normalized to those of β-actin. After amplification, PCR products were run on a 2% agarose gel in 90 mM Trisborate, 2 mM EDTA buffer (TBE), pH 8, and stained with ethidium bromide and observed by UV transillumination. To check the size of amplification products, a DNA ladder molecular weight marker (Gel Pilot 100 bp ladder (Cat. No. 239035) supplied by (QIAGEN, USA) was employed. Consequently, the product densities were analyzed by the gel documentation system (Bio Doc Analyze, Biometra, Germany) and photographs were taken using a Sony XC-75 CE camera (VilberLourant Inc. Cedex, France), with the density of the bands assessed using Photo-Capt v.99 Image software (VilberLourant Inc. Cedex, France).
2.5. Immunological and biochemical parameter evaluation
At the end of the feeding experiment, ten randomly selected fish from each group were collected and sedated with tricaine methane sulfonate MS-222 (100 ppm) for caudal vein blood sampling. Each blood sample was divided into two aliquots. One aliquot was placed in EDTA-containing tubes and was utilized for haematological analysis according to Feldman et al., (2000), and the second aliquot was allowed to clot at 4ᴼC and centrifuged at 1500 x g to separate the sera, which were then aliquoted and stored at -20ᴼC for ELISA assay. IL-1 and IL-6 cytokines, as well as immunoglobulin (IgM) levels in the various test groups, were measured, as well as some biochemical parameters. Both ELISA and biochemical assays were carried out as directed by the kits' manufacturer.
2.6. Tissue sampling
Intestinal and splenic tissues were obtained before and after the catfish treatments with the tested compounds in the four groups. Splenic tissues were employed for both histopathological and molecular detection of the target inflammatory cytokines' gene expression. Intestinal tissues were used for histopathological investigations.
2.7. Histopathological investigations
Specimens from the intestine and spleen were collected and immediately fixed in a 10% buffered neutral formalin solution for 48 hrs, then processed histologically, where the specimens were dehydrated in ascending grades of ethanol (70-100%), cleared in xylene, and finally embedded in paraffin. Five-micron thick paraffin sections were prepared and then routinely stained with Hematoxylin and Eosin (H&E) dyes for any histological changes according to [28]. The microphotographs were taken using a digital Dsc-W 130 super steady cyper shot camera (Sony, Japan) connected to an Olympus BX 21 light microscope.
2.8. Statistical analysis
SPSS version 20 was used to perform statistical analysis on the data. Statistical packages (IBM 1, New Orchard Road, Armonk, New York 10504-1722, United States) presented as a mean ±SD, n = 10 were used. Statistical differences among groups were performed using a one-way analysis of variance (ANOVA). Duncan’s test was used to test inter-group homogeneity. The level of statistical significance was set at p ≤ 0.05.